Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector.

Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1- kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (±931.7)- and 295,400 (±75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (±493,700)- and 308,000 (±139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/µg protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line.

DNA repair modulates the vulnerability of the developing brain to alkylating agents.

Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag(-/-)) or O(6)-methylguanine methyltransferase (Mgmt(-/-)), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt(-/-) neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag(-/-) neurons were for the most part significantly less sensitive than wild type or Mgmt(-/-) neurons to MAM and HN2. Aag(-/-) neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt(-/-) mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM-treated Aag(-/-) or MGMT-overexpressing (Mgmt(Tg+)) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in Mgmt(Tg+) mice treated with HN2. Collectively, these in vitro and in vivo studies demonstrate that the type of DNA lesion and the efficiency of DNA repair are two important factors that determine the vulnerability of the developing brain to long-term injury by a genotoxicant.

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.

Time-dependent effect of non-Hodgkin's lymphoma grade on disease-free survival of relapsed/refractory patients treated with high-dose chemotherapy plus autotransplantation.

Evaluation of time to event outcomes usually is examined by the Kaplan-Meier method and Cox proportional hazards models. We developed a modified statistical model based on histologic grade and other variables to describe the time-dependent outcome for autologous stem cell transplant (autotransplant) performed for non-Hodgkin's lymphoma (NHL) based on histologic grade and other variables. One hundred and fourteen relapsed or refractory NHL patients were treated using BCNU 600 mg/m2, etoposide 2400 mg/m2, and cisplatin 200 mg/m2 IV followed by autotransplant. Median age was 53.5 (range: 25-70) years, 78 patients had aggressive NHL and 36 indolent NHL. Seventy-five patients received involved-field radiotherapy just prior to transplant. At a median follow-up of 33 (range: 3 to 118) months, the estimated 5-year Kaplan-Meier probabilities of overall survival and disease-free survival were 61% and 51%, respectively. Cox proportional hazards model analysis showed that proportionality did not hold for lymphoma grade, indicating that the relationship between the grade and disease-free survival differed over time. By piece-wise Cox model, the relative risk for experiencing relapse or death after 1 year in patients with indolent compared with patients with aggressive NHL was 2.81 (p=0.019) with 95% confidence interval (1.19, 6.65). The time-dependent effect of lymphoma grade on disease-free survival suggests the need for early (within first year) incorporation of novel therapeutic approaches in management of patients with indolent NHL undergoing autotransplant.

Viral infection of vascular endothelial cells alters production of colony-stimulating activity.

Viral infections in humans are frequently associated with granulocytopenia and/or granulocytosis. Such changes in myelopoiesis could result from infection of the granulocyte-macrophage colony-forming cell (CFC-GM) or changes in the production of colony-stimulating activity (CSA). Endothelial cells are a known source of CSA and may be transiently or persistently infected during a number of viral infections, including infection with herpes simplex virus type I (HSV-I) and measles virus. Therefore, we examined the effect of endothelial cell infection with these two viruses on the production of CSA. Uninfected passaged endothelial cells produce CSA when stimulated by the continual presence of a factor present in medium conditioned by peripheral blood monocytes (MCM). Within 4 h of infection with HSV-I, endothelial cells no longer produced CSA in response to MCM. In contrast, measles virus infection induced CSA production by passaged endothelial cells even in the absence of MCM. Measles virus-induced CSA production was maximal at 24 h and required the presence of live virus within the endothelial cells. The effects of HSV-I and measles virus on CSA production were not dependent on alterations in the production of alpha- or gamma-interferon by the infected endothelial cells. Infection with HSV-I did not stimulate endothelial cells to release any detectable interferon. In contrast, the supernatants of the measles-infected cells contained only beta-interferon, a known inhibitor of CFC-GM development. These studies suggest that CSA production by endothelial cells is directly altered by infection with HSV-I and measles virus. An alteration in CSA production might contribute to changes in myelopoiesis that frequently accompany viral infection in humans.

O6 alkylguanine-DNA alkyltransferase activity in human myeloid cells.

The association between alkylating agent exposure and acute nonlymphocytic leukemia in humans indicates that myeloid cells may be particularly susceptible to mutagenic damage. Alkylating agent mutagenesis is frequently mediated through formation and persistence of a particular DNA base adduct, O6alkylguanine, which preferentially mispairs with thymine rather than cytosine, leading to point mutations. O6alkylguanine is repaired by O6alkylguanine-DNA alkyltransferase (alkyltransferase), a protein that removes the adduct, leaving an intact guanine base in DNA. We measured alkyltransferase activity in myeloid precursors and compared it with levels in other cells and tissues. In peripheral blood granulocytes, monocytes, T lymphocytes, and B lymphocytes, there was an eightfold range of activity between individuals but only a twofold range in the mean activity between cell types. Normal donors maintained stable levels of alkyltransferase activity over time. In bone marrow T lymphocytes and myeloid precursors, there was an eightfold range of alkyltransferase activity between donors. Alkyltransferase activity in the two cell types was closely correlated in individual donors, r = 0.69, P less than 0.005, but was significantly higher in the T lymphocytes than the myeloid precursors, P less than 0.05. Liver contained the highest levels of alkyltransferase of all tissues tested. By comparison, small intestine contained 34%, colon 14%, T lymphocytes 11%, brain 11%, and myeloid precursors 6.6% of the activity found in liver. Thus, human myeloid precursors have low levels of O6alkylguanine-DNA alkyltransferase compared with other tissues. Low levels of this DNA repair protein may increase the susceptibility of myeloid precursors to malignant transformation after exposure to certain alkylating agents.

Impaired growth and elevated fas receptor expression in PIGA(+) stem cells in primary paroxysmal nocturnal hemoglobinuria.

The genetic defect underlying paroxysmal nocturnal hemoglobinuria (PNH) has been shown to reside in PIGA, a gene that encodes an element required for the first step in glycophosphatidylinositol anchor assembly. Why PIGA-mutated cells are able to expand in PNH marrow, however, is as yet unclear. To address this question, we compared the growth of affected CD59(-)CD34(+) and unaffected CD59(+)CD34(+) cells from patients with that of normal CD59(+)CD34(+) cells in liquid culture. One hundred FACS-sorted cells were added per well into microtiter plates, and after 11 days at 37 degrees C the progeny were counted and were analyzed for their differentiation pattern. We found that CD59(-)CD34(+) cells from PNH patients proliferated to levels approaching those of normal cells, but that CD59(+)CD34(+) cells from the patients gave rise to 20- to 140-fold fewer cells. Prior to sorting, the patients' CD59(-) and CD59(+)CD34(+) cells were equivalent with respect to early differentiation markers, and following culture, the CD45 differentiation patterns were identical to those of control CD34(+) cells. Further analyses of the unsorted CD59(+)CD34(+) population, however, showed elevated levels of Fas receptor. Addition of agonist anti-Fas mAb to cultures reduced the CD59(+)CD34(+) cell yield by up to 78% but had a minimal effect on the CD59(-)CD34(+) cells, whereas antagonist anti-Fas mAb enhanced the yield by up to 250%. These results suggest that expansion of PIGA-mutated cells in PNH marrow is due to a growth defect in nonmutated cells, and that greater susceptibility to apoptosis is one factor involved in the growth impairment.

Pre-mobilization therapy blood CD34+ cell count predicts the likelihood of successful hematopoietic stem cell mobilization.

We examined pre-mobilization blood CD34+ cell count to predict ability to mobilize adequate peripheral blood progenitor cells (PBPC) in 106 cancer patients and 36 allogeneic donors. Mean pre-mobilization therapy blood CD34+ cell count was 3.1 cells x 10(6)/l (s.d. = 3.9, r = 0.3-37) and mean CD34+ cells collected were 5.3 x 10(6) cells/kg/leukapheresis procedure (s.d. = 7.0, r = 0.03-53). Yields correlated with pre-mobilization CD34+ cells x 10(6)/l (r = 0.37, P-value < 0.0001); correlation was stronger in allogeneic donors (r = 0.56, P-value = 0.0004) and males (r = 0.46, P-value < 0.0001). Based on classification and regression tree multivariate analysis, the predictive value of pre-mobilization blood CD34+ cell count was confounded by other variables, including age, gender, mobilization regimen and malignancy type. We generated an algorithm to predict a minimum PBPC yield of 1 x 10(6) CD34+ cells/kg/leukapheresis procedure after mobilization. A threshold pre-mobilization blood CD34+ cell count of 2.65 cells x 10(6)/l was the most important decision point in predicting successful mobilization. Only 2% of subjects with pre-mobilization blood CD34+ cell counts > 2.65 cells x 10(6)/l did not achieve the minimum per apheresis, whereas 24% with pre-mobilization values below threshold had inadequate mobilization. Prospectively identifying individuals at risk for mobilization failure would allow for improved treatment planning, resource utilization and time saving.

Regeneration of O6-alkylguanine-DNA alkyltransferase in human lymphocytes after nitrosourea exposure.

Mitogen-stimulated human lymphocytes have an increased capacity to repair many forms of DNA damage caused by UV, ionizing radiation, and chemical carcinogens. Human lymphocytes rely on a particular DNA repair protein, O6-alkylguanine-DNA alkyltransferase (alkyltransferase) to repair efficiently O6-alkylguanine, an important mutagenic adduct formed by nitrosoureas and other N-nitroso compounds. The alkyltransferase is a "suicide" protein which becomes inactivated during the repair process. Thus, basal activity and the ability to synthesize new protein activity are important compounds of O6-alkylguanine repair. We compared basal and regenerated alkyltransferase activity in resting and mitogen-stimulated human lymphocytes. During stimulation with L-phytohemagglutinin, alkyltransferase activity increased by a mean of 70% over resting cells. Following exposure to N-nitroso-N-methylurea (MNU), alkyltransferase activity was consumed in a dose-dependent manner in both resting and L-phytohemagglutinin-stimulated cells by the repair of MNU-induced O6-methylguanine-DNA adducts. Recovery of alkyltransferase activity began within 1 day of MNU exposure in the L-phytohemagglutinin-stimulated lymphocytes but did not occur in resting cells. Enzyme induction was not observed. When the alkyltransferase was only partially inactivated by low dose MNU, resting lymphocytes still failed to recover alkyltransferase activity. The rate of recovery of alkyltransferase activity in proliferating cells was dependent on the basal level of activity, which varied about 3-fold among donors. These data indicate that mitogen-stimulated lymphocytes develop an increased capacity to repair nitrosourea-induced DNA damage and are able to regenerate activity following nitrosourea exposure. In contrast, resting lymphocytes do not rapidly synthesize new alkyltransferase molecules after nitrosourea exposure and appear susceptible to DNA damage caused by persistent O6-alkylguanine adducts. Thus, both basal alkyltransferase activity and the proliferative state of normal lymphocytes influence the response to nitrosourea exposure.

Modulation of human lymphocyte O6-alkylguanine-DNA alkyltransferase by streptozotocin in vivo.

The ability to modulate DNA repair has been proposed as an effective method to overcome cytotoxic drug resistance in human tumors. However, no studies have shown that it is possible to achieve modulation of DNA repair in humans in vivo. This study analyzes modulation of O6-alkylguanine-DNA alkyltransferase, a DNA repair protein that protects cells from cytotoxic DNA adducts formed by nitrosoureas. Streptozotocin has been shown to inactivate the alkyltransferase in vitro and sensitize tumor cells to other nitrosoureas. Thus, we determined whether biochemical modulation of alkyltransferase activity could be documented in patients receiving therapeutic doses of streptozotocin and whether the modulation was specific to streptozotocin or occurred in patients undergoing treatment with other DNA-damaging agents as well. Normal peripheral blood lymphocytes were used to analyze modulation of the alkyltransferase. We found that lymphocyte alkyltransferase activity was significantly decreased 20 h after treatment with streptozotocin (500 mg/m2) or high dose 1,3-bis-(2-chloroethyl)-1-nitrosourea (350 mg/m2) but not after treatment with the other DNA-damaging agents or lower doses of 1,3-bis-(2-chloroethyl)-1-nitrosourea. A cumulative decline in lymphocyte alkyltransferase activity occurred with daily streptozotocin treatment, reaching 26 +/- 9% of control after the third day of treatment (P less than 0.0005). Thus, the alkyltransferase DNA repair protein can be modulated in vivo in humans given systemic drug treatment. While further studies are needed to document that biochemical modulation can be achieved in the target tumor in humans, this study supports the development of clinical trials using streptozotocin as a biochemical modulator of nitrosourea resistance in human malignancies.

The role of O6-alkylguanine DNA alkyltransferase in limiting nitrosourea-induced sister chromatid exchanges in proliferating human lymphocytes.

Although induction of sister chromatid exchanges (SCEs) following nitrosourea exposure may be greater during cell proliferation, the increase could be offset by the action of the DNA repair protein O6-alkylguanine DNA alkyltransferase (alkyltransferase). To evaluate these factors in resting and proliferating (phytohemagglutinin stimulated) human lymphocytes, we studied the effect of changes in alkyltransferase activity on 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-induced SCEs. Phytohemagglutinin stimulation resulted in induction of alkyltransferase activity (5.9 +/- 0.3 units, resting, versus 9.2 +/- 0.2 units, proliferating). In both resting and proliferating lymphocytes the alkyltransferase activity was inactivated by 85-88% after an 18-h exposure to 0.5 mM of the modified base O6methylguanine (O6mGua). However, 48 h after removal of O6mGua, proliferating lymphocytes recovered alkyltransferase activity while resting cells did not. In the absence of O6mGua, both resting and proliferating lymphocytes were equally sensitive to BCNU-induced SCEs. Following inactivation of the alkyltransferase by O6mGua, BCNU-induced SCEs were markedly increased, but the increase was much greater in resting than proliferating cells, 4-fold vs. 2.6-fold at each dose of BCNU (P less than 0.001). The factors providing partial protection against BCNU-induced SCEs in proliferating lymphocytes appear to include the proliferation-dependent increase in alkyltransferase activity and the ability of proliferating lymphocytes to rapidly recover alkyltransferase activity after its inactivation. Thus, the alkyltransferase appears to provide an important mechanism of resistance to SCE induction in human lymphocytes.

Potentiation of nitrosourea cytotoxicity in human leukemic cells by inactivation of O6-alkylguanine-DNA alkyltransferase.

The HL-60 promyelocytic leukemia cell line is resistant to nitrosoureas and contains high levels of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (alkyltransferase). We examined the protective role of the alkyltransferase in the nitrosourea resistance observed in this myeloid leukemia cell line to determine whether inactivation of the alkyltransferase with the modified base, O6-methylguanine (O6mGua), could sensitize these cells to nitrosoureas. The HL-60 cells were sensitized approximately 3.0-fold to five different nitrosoureas when the alkyltransferase was inactivated by 88% following a 24-h preincubation in 0.5 mM O6mGua. No effect of O6mGua preincubation was observed in the K562 chronic myelogenous leukemia cell line which is sensitive to nitrosoureas and has low levels of alkyltransferase activity. When regeneration of HL-60 alkyltransferase activity after exposure to nitrosoureas was prevented by maintaining cells in O6mGua, HL-60 became even more sensitive (3.7- to 8.5-fold) to nitrosoureas but remained slightly more resistant than K562. Next, we compared the dose of methyl- and chloroethylnitrosoureas which were cytotoxic in HL-60 with the dose which caused repair-induced inactivation of the alkyltransferase. Both methyl- and chloroethyl-nitrosoureas caused the dose-dependent inactivation of the alkyltransferase and with both, cytotoxicity was increased with O6mGua exposure. However, chloroethylnitrosoureas, which form a variety of O6 alkylation adducts, some of which are poorly repaired, exhibited 7-12 times more cytotoxicity relative to repair-induced inactivation of the alkyltransferase whereas methylnitrosoureas became cytotoxic only when the alkyltransferase had been inactivated. These data suggest that leukemic cells are sensitized to both methyl- and chloroethylnitrosoureas when O6mGua is used to persistently inactivate the alkyltransferase. However, the alkyltransferase provides more efficient protection from methylnitrosoureas than chloroethylnitrosoureas most likely because the latter form adducts which are poorly repaired by the protein and which if unrepaired may become cytotoxic cross-links.

Mismatch repair mutations override alkyltransferase in conferring resistance to temozolomide but not to 1,3-bis(2-chloroethyl)nitrosourea.

Cells with the mutator phenotype are tolerant to methylating damage from N-methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanine, exhibit replication repair errors, and have recently been found to be mutant in mismatch repair (MMR). However, resistance of cell lines with these defects to clinically used chemotherapeutic agents and the relationship of this resistance to expression of O6-alkylguanine-DNA alkyltransferase (AGT), which repairs DNA damage caused by methylating agents, has not been demonstrated. We compared resistance to the methylating agent temozolomide (TMZ) and to the chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), with and without AGT inhibition by 06-bG in several colorectal carcinoma cell lines. Two cell lines had known microsatellite instability (replication repair error-positive) and high levels of AGT, as well as a mutation in one of two MMR genes, hMLH1 (HCT116) or GTBP (HCT15). Cell line SW480 had wild-type MMR genes and high AGT, and HCT116+Ch3 has previously been transduced with chromosome 3 (carrying wild-type hMLH1) and thus has a "corrected" MMR phenotype. SW480 exhibited the expected sensitivity to TMZ and BCNU and marked potentiation of cytotoxicity by O6-bG. In contrast, HCT15 and HCT116 were markedly resistant to TMZ and were not sensitized by O6-bG-mediated inhibition of AGT, whereas the sensitivity pattern in HCT116+Ch3 cells was similar to that in SW480. All cell lines were sensitized to BCNU by O6-bG. Thus, tumor cells with defects in MMR appear particularly resistant to methylating agents in a manner that overrides dependence on AGT and its inhibition by O6-bG. However, these cells use AGT for resistance to chloroethylating agents, providing an alternative strategy for alkylating agent therapy.

Differential sensitivity of human and mouse alkyltransferase to O6-benzylguanine using a transgenic model.

O6-benzlguanine (O6-bG) potentiates nitrosourea cytotoxicity of human tumor xenografts in nude mice by inactivating O6-alkylguanine-DNA alkyltransferase (AGT). Recent reports dispute whether murine AGT, in cell-free systems, is less sensitive to O6-bG than the human AGT protein, raising the possibility that efficacy seen in the mouse host may not predict the therapeutic index observed in clinical trials. To establish whether mouse and human AGT have different sensitivity to O(6)-bG, we evaluated in vitro and in vivo models of O(6)-methylguanine-DNA methyltransferase gene (MGMT) expression in the same genetic background. The 50% inhibitory concentration of O6-bG for inactivation of mouse AGT was >10-fold higher than for the human protein in MGMT-transfected Chinese hamster ovary (CHO) cells. A dose of O6-bG, which inactivated human AGT, markedly sensitized human MGMT-transfected CHO cells to 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), whereas mouse MGMT-transfected CHO cells were much more resistant. O6-bG inactivation of AGT in vivo was studied in livers of human MGMT-transgenic mice expressing both human and mouse AGT. After a single dose of O6-bG i.p., the 50% inhibitory concentration of AGT was higher for mouse than for human AGT. To reconcile our finding with those of others, we sequenced the mouse MGMT cDNA and found that mutation of amino acid residue Leu180 was associated with O6-bG resistance. These studies provide strong evidence that inactivation of AGT both in vivo and in vitro by O6-bG is species selective and impacts O6-bG-mediated enhancement of BCNU toxicity. This may influence the therapeutic index of O6-bG-BCNU combinations observed in human tumor xenograft-bearing mice.

Repair of O6-alkylguanine during DNA synthesis in murine bone marrow hematopoietic precursors.

O6-Alkylguanine, a DNA adduct formed by nitrosoureas, becomes the site of a point mutation during DNA synthesis by preferentially base mispairing with thymine rather than correctly base pairing with cytosine. To repair this adduct, cells contain a limited amount of O6-alkylguanine-DNA alkyltransferase (alkyltransferase), a protein which removes the alkyl group in a stoichiometric reaction. To prevent mutations, repair must occur before DNA replication takes place. Consequently, formation of point mutations is related inversely to the number of alkyltransferase molecules and directly to the rate of DNA synthesis. Bone marrow hematopoietic precursors, the target for nitrosourea-induced leukemia, are deficient in alkyltransferase activity. We questioned whether regenerating bone marrow is more susceptible to nitrosoureas than other organs due to persistently low levels of alkyltransferase activity during periods of increased cell proliferation and DNA synthesis. Following syngeneic bone marrow transplantation, murine hematopoietic cells underwent rapid cell proliferation but alkyltransferase activity remained well below the activity in liver. After N-nitrosomethylurea exposure, [3H]thymidine incorporation in rat bone marrow increased 3-fold and stem cell proliferation over 10-fold within 2 days of exposure, but alkyltransferase activity remained low. The relative susceptibility of bone marrow to mutagenic damage from O6-alkylguanine adducts was determined by comparing the ratio of alkyltransferase activity to [3H]thymidine incorporation in marrow, kidney, and liver. In untreated animals, the ratio was lowest in bone marrow and decreased further 48 h after N-nitrosomethylurea exposure to only 21% that of kidney and 1% that of liver. Thus, proliferating hematopoietic precursors appear more likely to form point mutations following nitrosourea exposure than other rodent tissues because they undergo rapid proliferation soon after DNA damage and before O6-alkylguanine adducts can be repaired. The combination of rapid cell proliferation and low DNA repair capacity may be the mechanism of nitrosourea induced leukemic transformation of the bone marrow.

Modulation of O6-alkylguanine-DNA alkyltransferase in rats following intravenous administration of O6-methylguanine.

The purine analogue O6-methylguanine is an effective biochemical modulator of the DNA repair protein O6-alkylguanine-DNA alkyltransferase. Inactivation of the alkyltransferase by O6-methylguanine sensitizes tumor cells to nitrosoureas in vitro. The pharmacokinetics of O6-methylguanine were evaluated in female Sprague-Dawley rats following administration of a single 40 mg/kg i.v. bolus dose. Two-compartment pharmacokinetic analysis revealed a terminal elimination half-life of 2.3 +/- 0.68 h, a total body clearance of 6.0 +/- 0.53 ml/min/kg, and a volume of distribution at steady state of 948 +/- 186 ml/kg. To inactivate the alkyltransferase, 80 mg/kg O6-methylguanine was given at 0 and 2 h. Alkyltransferase decreased in bone marrow, kidney, lung, spleen, and intestine by 20-90%. Regeneration of alkyltransferase activity was observed 22-70 h after the first bolus dose of O6-methylguanine. A continuous infusion protocol, which achieved a steady state serum concentration of 10.3 +/- 1.5 micrograms O6-methylguanine/ml at 15 h, resulted in a similar degree of inactivation of tissue alkyltransferase to that observed following bolus drug infusion. O6-Methylguanine tissue concentrations, including that determined in brain, were 1.7- to 4-fold higher than that in serum, indicating that O6-methylguanine is concentrated in most if not all tissues. These studies establish pharmacokinetic parameters of O6-methylguanine in rats and suggest that effective biochemical modulation of alkyltransferase can be achieved in vivo. Further studies are indicated to assess the extent to which biochemical modulation of alkyltransferase reduces tumor nitrosourea resistance in vivo.

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.

Selection for G156A O6-methylguanine DNA methyltransferase gene-transduced hematopoietic progenitors and protection from lethality in mice treated with O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea.

A retroviral gene therapy approach was developed to protect early hematopoietic progenitors from 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a stem cell toxin, and O6-benzylguanine (BG), an inhibitor of a key BCNU resistance protein, O6-alkylguanine DNA alkyltransferase (AGT). The retroviral vector MFG was used to transfer the G156A MGMT (deltaMGMT) cDNA, encoding a mutant AGT that is resistant to inhibition by BG, into murine bone marrow-derived hematopoietic progenitors. Following transplantation into lethally irradiated mice, the transduced cells were subjected to in vivo BG and BCNU treatment to examine the ability to enrich for transduced cells expressing deltaAGT. Transplantation of deltaMGMT-transduced cells resulted in deltaAGT expression in 30% of bone marrow nucleated cells 13 weeks after transplantation. After one cycle of BG and BCNU, deltaAGT expression was observed in 60% of bone marrow cells, and the percentage of colony-forming units (culture; CFU-C) containing proviral sequence increased from 67 to 100%. CFU-C obtained from BG and BCNU-treated deltaMGMT animals up to 23 weeks after transplantation were more resistant to combination BG and BCNU than CFU-C from mice transplanted with lacZ-transduced cells and treated with BG and BCNU or from mice transplanted with deltaMGMT-transduced cells and left untreated. The degree of drug resistance in deltaMGMT-transduced hematopoietic progenitors to BG and BCNU was much greater than we observed previously with wild-type MGMT gene transfer and treatment with BCNU alone. Furthermore, whereas 21 of 22 mice transplanted with deltaMGMT-transduced cells survived in vivo BG and BCNU administration, only 3 of 13 mice transplanted with lacZ-transduced progenitors survived similar drug treatment. Thus, deltaMGMT-transduced murine bone marrow cells selectively survive in vivo BG and BCNU exposure, resulting in prolonged enrichment for the transduced cells and protection from mortality induced by this drug combination.

Eradication of neuroblastoma cells in vitro by monoclonal antibody and human complement: method for purging autologous bone marrow.

We describe an in vitro method which is useful for purging autologous bone marrow of neuroblastoma cells. The method utilizes a single murine monoclonal antibody 3G6 (an immunoglobulin MK) which we have previously developed against the ganglioside GD2; undiluted human complement; and unfractionated whole bone marrow at 1 X 10(7) nucleated cells/ml. Tumor cell clonogenic assays, Hoechst 33342 fluorescent nuclear stain, and trypan blue viability stain methods were used to assay cytotoxicity. This complement-mediated cytotoxicity technique killed 99.9-100% of neuroblastoma cell lines NMB-7, LAN-1, LAN-5, and IMR-6, while normal marrow precursor cells were not detectably damaged. The presence of normal bone marrow did not inhibit the human complement-mediated cytotoxicity. Applying the cytotoxicity method to whole unseparated bone marrow demonstrated killing of seeded neuroblastoma cells, with no gross hemolysis or cell clumping. The method did not require expensive special equipment, use of animal complement sera, or prior fractionation of the bone marrow. The average marrow nucleated cell recovery was 95%. These studies indicate that in vitro purging of autologous marrow infiltrated with neuroblastoma with monoclonal antibody 3G6 and human complement is both technically feasible and effective in eradicating residual tumor while preserving bone marrow stem cells.