Cloning and expression of canine O6-methylguanine-DNA methyltransferase in target cells, using gammaretroviral and lentiviral vectors

The human O(6)-methylguanine-DNA methyltransferase (MGMT) gene and its mutants have been used for in vivo selection of transduced hematopoietic stem cells with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) alone or in combination with O(6)-benzylguanine (BG). To allow similar in vivo selection in dogs, without the risk of inducing an immune response, we have cloned the canine MGMT drug resistance gene. Comparison of canine and human MGMT-coding regions indicates that there is about 62% amino acid identity and 78% similarity between the two MGMTs. The canine MGMT is also longer, by nine amino acids. Proline at position 140 and the surrounding amino acids of the human MGMT are highly conserved in the canine sequence. To determine whether mutation of the proline residue at position 144 to lysine in the canine MGMT would provide a similar advantage for selection of transduced cells as the human mutant, Moloney murine leukemia virus and human immunodeficiency type 1 vectors encoding the corresponding mutant MGMT were created and used to express separately canine and human MGMTs in cultured cells. Drug resistance assays using BCNU alone or BCNU with BG demonstrated that the wild-type and mutant canine MGMTs provided resistance to the selection agents that was comparable to the human MGMT counterparts.     

Characterization of the P140K, PVP(138-140)MLK, and G156A O6-methylguanine-DNA methyltransferase mutants: implications for drug resistance gene therapy

The G156A O6-alkylguanine-DNA alkyltransferase (AGT) mutant protein, encoded by the G156A O6-methylguanine-DNA methyltransferase gene (MGMT), is resistant to O6-benzylguanine (BG) inactivation and, after transduction into hematopoietic progenitors, transmits remarkable resistance to BG and BCNU. As a result, a clinical trial, in which the MGMT gene is transduced into CD34+ cells of patients with cancer, has been approved. A newly identified AGT mutation, P140K, generates dramatically increased BG resistance relative to G156A, and suggests that gene transfer of P140K may confer improved hematopoietic cell protection. To address this hypothesis, we measured BG + BCNU and BG + TMZ resistance in G156A, P140K, or P138M/V139L/P140K (MLK) MGMT-transduced K562 cells. In addition, we performed a detailed characterization of individual properties including BG resistance, activity, and protein stability of these mutants in human hematopoetic K562 cells and E86 retroviral producer cells. In K562 cell extracts, the MLK and P140K mutants retained full activity at doses up to 1 mM BG, while G156A had a BG ED50 of 15 microM, compared with 0.1 microM for wtAGT. In the absence of BG, the G156A protein possessed a 56% reduction in specific O6-methyltransferase activity compared with wtAGT. MLK, P140K, and wtAGT all possessed similar specific activities, although the O6-methyl repair rate of all mutants was reduced 4- to 13-fold relative to wtAGT. The wtAGT, MLK, and P140K proteins were stable, with half-lives of greater than 18 hr. In contrast, only 20% of the G156A protein was stable after 12 hr in cycloheximide and, interestingly, the remaining protein appeared to retain most of the activity present in non-cycloheximide-treated cells. Differences in BG resistance, activity, and stability between P140K, MLK, and G156A suggest that P140K may be the optimal mutant for drug resistance gene transfer. However, hematopoietic K562 cells transduced with MFG-G156A, P140K, or MLK had similar degrees of BG and BCNU as well as BG and TMZ resistance when treated with concentrations of BG (< or =25 microM) achieved in clinical trials, suggesting similar efficacy in many in vivo applications.     

Prolonged inhibition of O(6)-methylguanine DNA methyltransferase in human tumor cells by O(6)-benzylguanine in vitro and in vivo

We previously demonstrated that sustained depletion of methylguanine DNA methyltransferase (MGMT) activity is required for optimal reversal of chloroethylnitrosourea resistance in tumor cells. The purpose of this study was to design O(6)-benzylguanine (BG) treatments that deplete MGMT activity in tumor cells and xenograft tumors in a prolonged manner. When SF767 cells were treated with a bolus dose of BG (25 microM for 1 h), >95% of MGMT activity was depleted but 33% of the activity recovered within 24 h. In contrast, MGMT activity was completely depleted for 24 h when cells were pretreated with a low dose of BG (2.5 microM) for 24 h, followed by the bolus dose and same low-dose treatment for 24 h. This combination regimen of pre- and post-treatments with a bolus dose sensitized cells N,N'-bis(2-chloroethyl)-N-nitrosourea in vitro by approximately 2-fold more than the bolus dose alone. Similar BG treatment with Alzet micro-osmotic pumps produced sustained inhibition of MGMT activity in vivo. In xenograft SF767 tumors, low-dose pre- and post-treatments (8 mg/kg over 24 h) combined with an i.p. bolus dose (80 mg/kg) of BG inhibited >95% of MGMT activity for 24 h after the bolus. The bolus dose alone did not deplete MGMT for 24 h. These results demonstrate that combination low-dose and bolus BG treatment is superior to the bolus dose alone in depleting MGMT activity in a sustained manner in vitro and in vivo. When combined with N,N'-bis(2-chloroethyl)-N-nitrosourea treatment, this BG regimen also should also produce greater antitumor activity than the single bolus dose evaluated clinically.     

Cytotoxicity,DNA damage,and apoptosis induced by new fotemustine analogs on human melanoma cells in relation to O6-methylguanine DNA-methyltransferase expression

Fotemustine is a third generation chloroethylnitrosourea that has demonstrated significant antitumoral effects in malignant melanoma. However, its use is somewhat limited by its toxic side effects and chemoresistance caused by direct repair of O6-alkyl groups by the enzyme O6-methylguanine DNA-methyltransferase (MGMT). The aim of this work was to determine to what extent the expression of MGMT influences cytotoxicity, DNA damage, and apoptosis induced by new nitrososulfamide analogs of fotemustine (compounds 4 and 8), which have previously demonstrated interesting antiproliferative properties. We carried out complementary strategies that consisted of MGMT cDNA transfection in CAL77 Mer- melanoma cells and of MGMT inhibition with O6-benzylguanine (BG) in A375 Mer+ melanoma cells. MGMT-transfected cells were 7 to 9 times less sensitive to fotemustine than parent cells, whereas no difference between the transfected and parent cells was observed for nitrososulfamide analogs. The cytotoxicity of these analogs vis à vis a MGMT-proficient A375 melanoma cell line was approximately 3 times greater than that of fotemustine. Coincubation of these cells with O6-benzylguanine significantly increased the cytotoxicity of fotemustine and compound 8, whereas BG had little effect on the cytotoxicity of compound 4. Furthermore, DNA fragmentation determined by a comet assay was greater with nitrososulfamide analogs than with fotemustine. O6-benzylguanine increased DNA fragmentation for fotemustine and compound 8, but not for compound 4, which induced comets with a typical apoptotic appearance. The ability of this compound to induce apoptosis in the absence of BG was confirmed by a specific enzyme-linked immunosorbent assay apoptotic assay using a single-stranded DNA monoclonal antibody.     

Inhibition of acid secretion in guinea pigs by tricyclic antidepressants: comparison with ranitidine and omeprazole

The antisecretory properties of imipramine on gastric secretion in guinea pig in comparison with other antisecretory agents was determined. In awake guinea pigs s.c. infusion of histamine (30 micrograms/kg/hr) increased acid and fluid secretion by 3- to 4-fold. When acid output peaked, a bolus administration of the tricyclic anti-depressant imipramine inhibited acid and fluid secretion. Imipramine and other agents, such as ranitidine and omeprazole, inhibited gastric secretion in a dose-dependent fashion. The most potent was the H2-antagonist ranitidine (IC50, 0.2-0.3 mumol/kg), followed by the gastric H-K-adenosine triphosphatase inhibitor, omeprazole (IC50, 0.5-0.6 mumol/kg). Imipramine (IC50 1-2 mumol/kg) was the least potent of the inhibitors. Both ranitidine and omeprazole could abolish acid secretion, but maximal inhibition with imipramine was 60% of initial. Promethazine (25 mumol/kg), an H1 antagonist, and atropine (12 mumol/kg), a muscarinic antagonist, inhibited gastric secretion by 40 to 50%. Imipramine and atropine also inhibited basal acid secretion. In dispersed gastric cells comparison between imipramine and omeprazole showed that imipramine was about 5-fold more potent than omeprazole in blocking histamine or dibutyryl cyclic AMP stimulation of aminopyrine accumulation. Imipramine probably acts as a protonophore by increasing the rate of proton-gradient dissipation rather than by interfering with the hydrogen-pump system because, in gastric membranes, imipramine was 20-fold less potent than omeprazole in inhibiting the gastric H-K-adenosine triphosphatase activity. These results suggest that imipramine administered s.c. in guinea pigs is a potent antisecretory drug. Its action may be due to a combination of anticholinergic and antihistamine H2 activities.     

Inhibition of dynamin by dynole 34-2 induces cell death following cytokinesis failure in cancer cells

Inhibitors of mitotic proteins such as Aurora kinase and polo-like kinase have shown promise in preclinical or early clinical development for cancer treatment. We have reported that the MiTMAB class of dynamin small molecule inhibitors are new antimitotic agents with a novel mechanism of action, blocking cytokinesis. Here, we examined 5 of the most potent of a new series of dynamin GTPase inhibitors called dynoles. They all induced cytokinesis failure at the point of abscission, consistent with inhibition of dynamin while not affecting other cell cycle stages. All 5 dynoles inhibited cell proliferation (MTT and colony formation assays) in 11 cancer cell lines. The most potent GTPase inhibitor, dynole 34-2, also induced apoptosis, as revealed by cell blebbing, DNA fragmentation, and PARP cleavage. Cell death was induced specifically following cytokinesis failure, suggesting that dynole 34-2 selectively targets dividing cells. Dividing HeLa cells were more sensitive to the antiproliferative properties of all 5 dynoles compared with nondividing cells, and nontumorigenic fibroblasts were less sensitive to cell death induced by dynole 34-2. Thus, the dynoles are a second class of dynamin GTPase inhibitors, with dynole 34-2 as the lead compound, that are novel antimitotic compounds acting specifically at the abscission stage.     

Inhibition of rat liver estrogen 2/4-hydroxylase activity by troleandomycin: comparison with erythromycin and roxithromycin

Administration of troleandomycin (0.5 mmol.kg-1 p.o. daily for 5 days) decreased by 61% and 36%, respectively, the estradiol and ethinylestradiol 2/4-hydroxylase activities of hepatic microsomes from male Sprague-Dawley rats killed 2 hr after the last dose. This decrease did not appear to be due to the in vivo formation of the inactive cytochrome P-450 p Fe(II)-metabolite complex, since disruption of this complex with potassium ferricyanide did not increase estrogen hydroxylase activities. Troleandomycin administration, however, essentially suppressed cytochrome P-450 UT-A (one of the P-450 forms involved in the hydroxylation of estrogens) and resulted in the appearance of cytochrome P-450 forms whose estradiol hydroxylase activity was inhibitable by troleandomycin in vitro. Similarly, troleandomycin (2 mM) inhibited by 60% estradiol and ethinylestradiol 2/4-hydroxylase activities in microsomes from dexamethasone-treated rats, although it had no inhibitory effect in microsomes from control rats. In contrast, erythromycin and roxithromycin (2 mM) exerted no inhibitory effect, even in microsomes from dexamethasone-treated rats. In vivo, these macrolides (0.5 mmol.kg-1 p.o. daily for 5 days) decreased moderately cytochrome P-450 UT-A levels and estradiol 2/4-hydroxylase activity, and did not modify ethinylestradiol 2/4-hydroxylase activity. We conclude that the administration of troleandomycin, but not that of erythromycin or roxithromycin, decreases ethinylestradiol 2/4-hydroxylase activity in male rat liver microsomes, as a possible consequence of decreased cytochrome P-450 UT-A levels and of the induction of glucocorticoid-responsive P-450 forms whose ethinylestradiol hydroxylase activity is inhibitable by troleandomycin.     

Photodynamic sterilization of red cells and its effect on contaminating white cells: viability and mechanism of cell death

BACKGROUND: Phthalocyanines are useful sensitizers for photodynamic sterilization of red cell concentrates. Various lipid-enveloped viruses can be inactivated with only limited red cell damage. Because white cells are involved in the immunomodulatory effects of blood transfusions, the study of the effect of photodynamic treatment on these cells is imperative. STUDY DESIGN AND METHODS: White cell-enriched red cell suspensions were photodynamically treated with either the hydrophobic Pc4 (HOSiPcOSi-(CH3)2(CH2)3N(CH3)2) or water-soluble aluminum phthalocyanine tetrasulfonate (AIPCS4) under virucidal conditions. Viability of white cell subpopulations on Days 0, 1, and 4 after treatment was determined by fluorescence-activated cell sorting by flow cytometric analysis of propidium iodide uptake. Apoptosis induction was studied by DNA ladder formation and staining for an early marker of apoptosis (annexin V). RESULTS: Treatment with Pc4 causes a significant decrease in cell viability of all white cells, as shown by prodidium iodide uptake. Monocytes and granulocytes are the most sensitive, and lymphocytes are relatively more resistant. Some of the cells die by apoptosis, which is induced within 30 minutes after treatment. Treatment with AIPCS4 damages only monocytes; other cell populations are not affected. CONCLUSIONS: Physicochemical properties of the photosensitizers partly determine their effect on white cells. Differences in intracellular localization are likely to be responsible for the effects observed.     

Antitumor effect on murine renal cell carcinoma by autologous tumor vaccines genetically modified with granulocyte-macrophage colony-stimulating factor and interleukin-6 cells

The authors evaluted the efficacy of vaccination with murine renal cell carcinoma (Renca) secreting the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and interleukin-6 (IL-6) gene for the treatment of Renca tumor. Murine GM-CSF and murine IL-6 genes were introduced and expressed in Renca cells (Renca-GM-CSF and Renca-IL-6). For a prevaccination study, wild-type Renca cells were injected subcutaneously into Balb/c mice that had been vaccinated three times with inactivated wild-type Renca, Renca-GM-CSF, Renca-IL-6, or a mixture of Renca-GM-CSF and Renca-IL-6 cells 7, 14, and 21 days before this tumor inoculation. For vaccination experiments, Renca tumor-bearing (8 to 10 mm) mice were injected subcutaneously weekly for 3 weeks with inactivated wild-type Renca cells, or either one or a combination of Renca-GM-CSF and Renca-IL-6. A nonvaccinated control was included in all experiments. The animals were monitored for survival and tumor development for 8 weeks. Mice inoculated with wild-type Renca alone died from the tumor within 35 days. Renca-IL-6 grew slower than wild-type Renca (p < 0.05). No tumor was produced by Renca-GM-CSF. Prevaccination with the combination of Renca-GM-CSF and Renca-IL-6 prevented subsequently inoculated wild-type Renca from forming tumors, and prevaccination with either one of them, compared with prevaccination with wild-type Renca, retarded tumor growth and prolonged survival time. Tumor-bearing mice vaccinated with wild-type Renca died within 42 days. Vaccination with Renca-GM-CSF or Renca-IL-6 alone prolonged the survival time, but only Renca-GM-CSF drastically reduced the tumor size. Vaccination with the combination of them achieved complete remission. Neither of the cytokine-secreting cells enhanced the expression of MHC class I or II molecules. Autologous tumor cell vaccine secreting GM-CSF is effective in preventing and treating established tumors. Its efficacy is enhanced by the cosecretion of IL-6.     

Inhibition of platelet-dependent thrombosis by low molecular weight heparin (CY222): comparison with standard heparin

We have compared the relative antithrombotic and antihemostatic effects of the very low molecular weight heparin CY222 with standard unfractionated heparin (SH) in a baboon model of platelet-dependent thrombosis. Thrombus formation was induced by placement of a thrombogenic device in an exteriorized femoral arteriovenous shunt under conditions of intermediate-shear blood flow. The device consisted of a collagen-coated cannular segment positioned proximal to two regions of expanded diameter exhibiting disturbed flow and stasis. Thrombus formation was measured in real time by indium 111-labeled platelet imaging. The collagen-coated surface accumulated thrombi composed largely of platelets, and the regions of disturbed flow were morphologically rich in fibrin and red cells. SH and CY222 were administered by continuous infusion for 1 hour. Although both heparin preparations abolished thrombus formation in the low-shear fibrin-rich regions at plasma levels less than 0.5 anti-Xa U/ml, platelet deposition onto the collagen surface was not reduced by either SH or CY222 at that dosage. These findings were consistent with previously observed therapeutic benefits of this level of anti-Xa activity in venous, but not arterial, thrombosis. Platelet deposition on the collagen was reduced in a dose-dependent manner by both SH and CY222 administered at doses between 1 and 5 anti-Xa U/ml. It is important to note that although heparin preparations produced profound and equivalent antithrombotic effects for platelet-dependent thrombus formation at comparable levels of anti-Xa activity, SH prolonged both the coagulation time and the bleeding time substantially more than did CY222.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of human non-small cell lung tumors by a c-Met antisense/U6 expression plasmid strategy

c-Met is a receptor tyrosine kinase whose activation by hepatocyte growth factor (HGF) can lead to transformation and tumorigenicity in a variety of tumors. We investigated the effects of suppressing c-Met protein expression in human non-small cell lung tumors. Expression plasmids containing either sense or antisense sequences of the human c-met gene were constructed under control of the U6 snRNA promoter. A U6 control plasmid was also constructed that did not contain any c-met sequence. These constructs have been examined both in vitro and in an in vivo tumor xenograft model. The c-Met protein was downregulated by 50-60% in two lung cancer cell lines that were transiently transfected with the c-Met antisense versus U6 control. Tumor cells treated with the c-Met antisense construct also show decreased phosphorylation of c-Met and MAP kinase when exposed to exogenous HGF. Lung cancer cells were grown as xenografts in mice and treated by intratumoral liposome-mediated transfer of the c-Met sense, antisense or U6 control plasmids. The treatment of lung tumors with c-Met antisense versus U6 control plasmid resulted in the downregulation of the c-Met protein expression, a 50% decrease in tumor growth over a 5-week treatment period and an increased rate of apoptosis. These results suggest that targeting the HGF/c-Met pathway may be an effective novel strategy to treat lung cancer patients.     

Quantitative effect of CYP2D6 genotype and inhibitors on tamoxifen metabolism: implication for optimization of breast cancer treatment

BACKGROUND AND OBJECTIVES: N-Desmethyltamoxifen (NDM), a major primary metabolite of tamoxifen, is hydroxylated by cytochrome P450 (CYP) 2D6 to yield endoxifen. Because of its high antiestrogenic potency, endoxifen may play an important role in the clinical activity of tamoxifen. We conducted a prospective trial in 158 patients with breast cancer who were taking tamoxifen to further understand the effect of CYP2D6 genotype and concomitant medications on endoxifen plasma concentrations. METHODS: Medication history, genotype for 33 CYP2D6 alleles, and plasma concentrations of tamoxifen and its metabolites were determined at the fourth month of tamoxifen treatment. RESULTS: By use of a mixture model approach, endoxifen plasma concentration identified 2 phenotypic groups, whereas 4 were defined by the endoxifen/NDM plasma concentration ratio. Three distinct genotype groups were identified in the distribution of endoxifen/NDM ratio: (1) low ratios composed of patients lacking any functional allele (mean, 0.04 +/- 0.02); (2) intermediate ratios represented by patients with 1 active allele (mean, 0.08 +/- 0.04); and (3) high ratios composed of patients with 2 or more functional alleles (mean, 0.15 +/- 0.09). Endoxifen/NDM plasma ratios were significantly different between these groups (P < .001). The mean endoxifen plasma concentration was significantly lower in CYP2D6 extensive metabolizers who were taking potent CYP2D6 inhibitors than in those who were not taking CYP2D6 inhibitors (23.5 +/- 9.5 nmol/L versus 84.1 +/- 39.4 nmol/L, P < .001). CONCLUSION: CYP2D6 genotype and concomitant potent CYP2D6 inhibitors are highly associated with endoxifen plasma concentration and may have an impact on the response to tamoxifen therapy. These iterative approaches may be valuable in the study of other complex genotype-phenotype relationships.     

Inhibition of cytotoxic T lymphocyte-induced target cell DNA fragmentation, but not lysis, by inhibitors of DNA topoisomerases I and II

Cytotoxic T lymphocytes (CTL) kill their target cells via a contact-dependent mechanism that results in the perturbation of the target cell's plasma membrane and the fragmentation of the target cell's DNA into nucleosomal particles. The membrane disruption is presumed to be due to the action of perforin, while the DNA fragmentation is thought to be by the activation of an endogenous nuclease(s). DNA topoisomerases I and II are nuclear enzymes with inherent endonuclease activities. We have investigated their role in the CTL-induced DNA fragmentation process. We report that in CTL killing assays, the treatment of target cells with topoisomerase I and II inhibitors blocks the CTL-induced DNA fragmentation process, but not the lysis of the target cell.     

Inhibition of cell proliferation and fibronectin biosynthesis by Na ascorbate

BACKGROUND: The importance of ascorbate on the production of extracellular matrix proteins (as elastin and collagens) is now well documented, but no studies have been published concerning its effects on fibronectin biosynthesis. Fibronectin is important for cell attachment and for proliferation. MATERIALS AND METHODS: The effects of Na ascorbate were investigated on cell attachment, proliferation, viability and fibronectin biosynthesis by human skin fibroblasts in vitro. Proliferation was followed by the monitoring of [(3)H]-thymidine incorporation; viability by the MTT-test, cell adherence by counting adherent and nonadherent cells and fibronectin biosynthesis by immunoprecipitation of biosynthetically labelled fibronectin. RESULTS: In the presence of ascorbate, the fibroblasts showed a biphasic growth pattern. At 500 microM ascorbate, [(3)H]-thymidine incorporation was stimulated by 15% as compared to the controls. Higher concentrations gradually decreased proliferation up to 36% of the control value at 5 mM. These effects of ascorbate on DNA synthesis were followed to > 1.25 mM by a strong inhibition, cytotoxic effect and cell death. The non-adherent cell count increased to 10% of the total population at 2.5 mM and to 31% at 5.0 mM ascorbate.Increasing concentrations of ascorbate resulted in a dose-dependent decrease of fibronectin biosynthesis, both in the culture supernates and cell extracts. This inhibition mainly concerned cell membrane-associated fibronectin.Superoxide-dismutase or catalase could inhibit Na ascorbate-induced cytotoxicity and partially re-establish fibronectin biosynthesis. Desferrioxamine, ergothionein and vitamin E were inefficient. CONCLUSIONS: Our results indicate that ascorbate decreases fibronectin biosynthesis of cultured human skin fibroblasts, thereby producing cell detachment and decreased proliferation. This effect is mainly mediated by the reactive oxygen species and can be inhibited by superoxide-dismutase and catalase.     

Differential effect of the protein synthesis inhibitors puromycin and cycloheximide on vascular smooth muscle cell viability

Recent evidence indicates that the protein synthesis inhibitor cycloheximide triggers selective macrophage death in rabbit atheroma-like lesions without affecting smooth muscle cells (SMCs) or the endothelium, thereby favoring a stable plaque phenotype. In this study, we report that puromycin, a protein synthesis inhibitor with a different mode of action but with similar ability to inhibit de novo protein synthesis, did not reveal plaque-stabilizing effects. The macrophage and the SMC content readily decreased in puromycin-treated atheroma-like lesions in rabbit carotid arteries. Moreover, puromycin induced apoptosis in macrophages and SMCs in vitro. Puromycin-treated SMCs showed signs of endoplasmic reticulum (ER) stress, as demonstrated by CCAAT/enhancer-binding protein homologous protein (CHOP) protein expression, splicing of X-box-binding protein 1 mRNA, and phosphorylation of eukaryotic translation initiation factor 2alpha. The ER stress inducer thapsigargin up-regulated CHOP protein expression in SMCs without affecting their viability, indicating that ER stress not necessarily results in cell death. Puromycin, but not thapsigargin, activated the ER stress-related caspase-12. Treatment of SMCs with a combination of cycloheximide and puromycin inhibited ER stress and partially improved SMC viability. In addition, puromycin, but not cycloheximide or thapsigargin, induced intracellular accumulation of polyubiquitinated proteins in SMCs, whereas the proteasome function was not affected. Taken together, puromycin, in contrast to cycloheximide, induces SMC apoptosis, thereby favoring an unstable plaque phenotype. SMC death upon puromycin treatment could only be partially prevented by cycloheximide, which completely blocked ER stress. However, other or additional mechanisms, such as increased polyubiquitination of proteins, might be involved in puromycin-induced SMC death.