Impact of NADPH oxidase functional polymorphisms in acute myeloid leukemia induction chemotherapy.

Efficacy and toxicity of anthracycline treatment in acute myeloid leukemia (AML) is mediated by reactive oxygen species (ROS). NADPH oxidase is the major endogenous source of ROS and a key mediator of oxidative cardiac damage. The impact of NADPH oxidase polymorphisms (CYBA:rs4673, NCF4:rs1883112, RAC2:rs13058338) was evaluated in 225 adult de novo AML patients. Variant alleles of NCF4 and RAC2 were related to higher complete remission (P=0.035, P=0.016), and CYBA homozygous variant showed lower overall survival with recessive model (P=0.045). Anthracycline-induced cardiotoxicity was associated to NCF4 homozygous variant (P=0.012) and CYBA heterozygous genotype (P=0.027). Novel associations were found between variant allele of CYBA and lower lung and gastrointestinal toxicities, and a protective effect in nephrotoxicity and RAC2 homozygous variant. Moreover, RAC2 homozygous variant was related to delayed thrombocytopenia recovery. This study supports the interest of NADPH oxidase polymorphisms regarding efficacy and toxicity of AML induction therapy, in a coherent integrated manner.

A systematic review and meta-analysis of the impact of WT1 polymorphism rs16754 in the effectiveness of standard chemotherapy in patients with acute myeloid leukemia.

The polymorphism rs16754 of the WT1 gene has been described as a possible prognostic marker in different acute myeloid leukemia (AML) cohorts; however, it is not supported by all the studies. We performed the first meta-analysis evaluating the effect of this polymorphism upon the effectiveness of standard AML therapy. Fourteen cohort studies were included (3618 patients). Patients with the variant allele showed a significant higher overall survival (OS) at 5 years (OR:1.24, 95% CI: 1.06-1.45, P=0.007, with dominant model). WT1 did not influence complete remission, but a higher disease-free survival was observed with the variant allele. In the subgroup analysis, Caucasians, pediatric and patients treated with idarubicin and etoposide carrying the variant allele showed consistent results in OS, whereas patients with cytogenetically normal AML did not show differences. To verify the effect of this polymorphism upon other outcomes, studies in larger and multiracial populations are needed.

Influence of ABCB1 polymorphisms upon the effectiveness of standard treatment for acute myeloid leukemia: a systematic review and meta-analysis of observational studies.

The ABCB1 gene encodes for P-glycoprotein (P-gp), an efflux pump for a variety of xenobiotics. The role of ABCB1 polymorphisms in acute myeloid leukemia (AML) outcomes of standard chemotherapy (cytarabine plus anthracyclines) remains controversial. A systematic search was made of studies evaluating the association between ABCB1 polymorphisms 1236C>T, 2677G>T/A and 3435C>T and effectiveness variables. We found seven cohort studies (1241 patients) showing a significantly higher overall survival (OS) among carriers of the variant allele of 1236C>T at year 4 (odds ratio (OR): 1.47, 95% confidence interval (CI): 1.07-2.01), 2677G>T/A at years 4-5 (OR: 1.37, 95% CI: 1.01-1.86) and 3435C>T at years 3 (OR: 1.41, 95% CI: 1.03-1.94) and 4-5 (OR: 1.42, 95% CI: 1.05-1.91). In the subgroup analysis according to ethnicity, Caucasians carrying variant allele showed consistent results in OS. ABCB1 influence upon complete remission could not be demonstrated. Future studies based on larger populations and multiethnic groups should help clarify the effect of P-gp polymorphisms upon other outcomes.

Effect of CYP3A5*3 on kidney transplant recipients treated with tacrolimus: a systematic review and meta-analysis of observational studies.

The highly variable pharmacokinetics of tacrolimus can hamper the optimal management of kidney transplant patients. This variability has been attributed to the genetic polymorphism of CYP3A5 6986A>G, but the evidence is not clear. We conducted a meta-analysis of studies evaluating the effect of CYP3A5 polymorphism on kidney transplant recipients with tacrolimus plasma concentration divided by daily dose per body weight (C/D) and clinical outcomes. We searched in MEDLINE and EMBASE. We found evidence suggesting a significantly lower C/D among CYP3A5*1 allele carriers compared with carriers of the CYP3A5*3/*3 genotype at weeks 1 and 2, and months 1, 3, 6 and 12. We demonstrated that the expresser genotype might have higher risk of acute rejection and chronic nephrotoxicity. In conclusion, CYP3A5 6986A>G polymorphism can affect tacrolimus pharmacokinetics and the incidence of acute rejection and chronic nephrotoxicity on kidney transplant recipients. Patients at high risk of developing tacrolimus-related complications could be detected even before their kidney transplant.

A surgical model for isolating the pig liver in vivo for gene therapy.

Several studies report results that suggest the need of vascularization blocking for efficient gene transfer to the liver, especially in nonviral gene therapy. In this study, we describe a surgical strategy for in vivo isolation of the pig liver, resulting in a vascular watertight organ that allows the evaluation of several gene injection conditions. The hepatic artery and portal, suprahepatic and infrahepatic cava veins were dissected. Then, liver vascularization was excluded for 5-7 min. In that time, we first injected 200 ml saline solution containing the p3c-eGFP plasmid (20 µg/ml) simultaneously through two different catheters placed in the portal and cava veins, respectively. Vital constants were monitored during the surgery to assess the safety of the procedure. Basal systolic/diastolic blood pressures were 92.8/63.2 mm Hg and dropped to 40.7/31.3 mm Hg at the end of vascular exclusion; the mean basal heart rate was 58 bpm, reaching 95 bpm when the blood pressure was low. Oxygen saturation was maintained above 98% during the intervention, and no relevant changes were observed in the ECG tracing. Peak plasma AST (aspartate aminotransferase) and ALT (alanine aminotransferase) levels were observed after 24 h (151 and 57 IU, respectively). These values were higher, but not relevant, in 60 ml/s injection than in 20 ml/s injection. Efficiency of gene transfer was studied with simultaneous (cava and portal veins) injection of eGFP gene at flow rates of 20 and 60 ml/s. Liver tissue samples were collected 24 h after injection and qPCR was carried out on each lobe sample. The results confirmed the efficiency of the procedure. Gene delivery differed between 20 ml/s (9.9-31.0 eGFP DNA copies/100 pg of total DNA) and 60 ml/s injections (0.6-1.1 eGFP DNA copies/100 pg of total DNA). Gene transcription showed no significant differences between 20 ml/s (15,701.8-21,475.8 eGFP RNA copies/100 ng of total RNA) and 60 ml/s (12,014-36,371 eGFP RNA copies/100 ng of total RNA). The procedure is not harmful for animals and it offers a wide range of gene delivery options because it allows different perfusion ways (anterograde and retrograde) and different flow rates to determine the optimal conditions of gene transfer. This strategy permits the use of cell therapy and viral or non-viral liver gene therapy, especially appropriated to a wide variety of inherited or acquired diseases because of the liver's ability to produce and deliver proteins to the bloodstream.

DNA delivery to 'ex vivo' human liver segments.

Hydrodynamic injection is an efficient procedure for liver gene therapy in rodents but with limited efficacy in large animals, using an 'in vivo' adapted regional hydrodynamic gene delivery system. We study the ability of this procedure to mediate gene delivery in human liver segments obtained by surgical resection. Watertight liver segments were retrogradely injected from hepatic vein with a saline solution containing a plasmid bearing the enhanced green fluorescent protein (eGFP) gene, under different conditions of flow rate (1, 10 and 20 ml s(-1)) and final perfused volume. Samples were cultured for 1 to 2 days and used for microscopy and molecular analysis of gene expression. The fluorescent and immunohistochemistry studies indicated that in segments injected at ≥10 ml s(-1), good and wide gene expression was present in the liver sections and the molecular analysis reinforced the histological observation in a quantitative manner (index of apparent gene delivery: 10(2)-10(4) eGFP DNA copy per 100 pg of total DNA; transcription index: 10(5)-2 × 10(6) eGFP RNA copy per 100 ng of total RNA). In addition, injected gold nanoparticles (15 nm diameter) suggested that DNA delivery to hepatocytes must involve a facilitated permeation process without membrane disruption. In summary, we show that retrograde venous injection of watertight human liver segment is an anadromous procedure that results in wide liver gene delivery and good gene expression. However, additional studies will be necessary to clarify the influence of the prolonged ischemia injury to hepatocytes in our model.

Real-time quantification of human telomerase reverse transcriptase mRNA in the plasma of patients with prostate cancer.

The aim of this study was to evaluate the potential diagnostic value of quantitative analysis of human telomerase reverse transcriptase (hTERT) mRNA in plasma for noninvasive diagnosis of prostate cancer (PCa). Expression levels of hTERT were analyzed by real-time quantitative RT-PCR in 68 patients showing elevated prostate-specific antigen (PSA) levels and a control group of 44 healthy volunteers. Sensitivity and specificity were determined and compared to the corresponding PSA values. Median values for hTERT gene expression in the PCa patients (0.72 ng; range 0.01-12.86) were statistically significantly higher (P < 0.001) than in the control group (0.13 ng; 0.02-0.35). Patients with clinically confirmed prostatitis showed lower plasma hTERT expression than PCa patients (0.29; 0.01-66.07). At a cutoff value of 0.35 sensitivity and specificity for the diagnosis of PCa were 81% and 60%, respectively. We suggest that hTERT mRNA in plasma is a very specific and sensitive method that may aid to differentiate between malignant and nonmalignant prostate tissue and may be a useful marker (in combination with PSA) for early PCa diagnosis.

Asialofetuin liposome-mediated human alpha1-antitrypsin gene transfer in vivo results in stationary long-term gene expression.

The development of nonviral vectors for in vivo gene delivery to hepatocytes is an interesting topic in view of their safety and tremendous gene therapy potential. Since cationic liposomes and liposome uptake by receptor-mediated mechanisms could offer advantages in the efficacy of liposome-mediated gene transfer, we studied the effect of liposome charge (anionic vs. cationic) and the covalently coupled asialofetuin ligand on the liposome surface in mediating human alpha1-antitrypsin (hAAT) gene transfer to mice in vivo. The changes in liposome charge were made by adding the following lipids to the backbone liposomes: anionic phosphatidylserine, cationic N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate or a lipopeptide synthesized from dipalmitoylphosphatidylethanolamine and covalently coupled to the cationic nuclear localization signal peptide. Two plasmids containing the hAAT gene were used: pTG7101, containing the complete genomic sequence of the human gene driven by the natural promoter, and p216, containing the human hAAT cDNA under the control of the CMV promoter. The results indicate that both untargeted anionic and cationic liposomes mediate plasma levels of hAAT that decline over time. However, asialofetuin liposomes increase the plasma levels of hAAT and can mediate long-term gene expression (>12 months) with stationary plasma levels of protein. Results from quantitative and qualitative reverse transcriptase polymerase chain reaction match those from protein plasma levels and confirm both the human origin of the message and the liver as source of the protein. The use of asialofetuin liposomes in hepatic gene therapy may both increase and prolong in vivo gene expression of hAAT and other clinically important genes.

Antisense gene therapy using anti-k-ras and antitelomerase oligonucleotides in colorectal cancer.

AIM: To test the efficacy of anti-k-ras and antitelomerase oligonucleotides for disabling colorectal cancer cell growth. MATERIAL AND METHODS: An established human colorectal cancer cell line (SW 480, ATTC) was used. Oligodeoxiribonucleotides (ODNs) have a phosphorotioate modification to ensure intracellular intake. We used an antitelomerase ODN (Telp5) and two anti-k-ras ODNs (AS-KRAS and ISIS). AS-KRAS is designed to join the k-ras oncogene s exon 1. ISIS links to the terminal transcription unit 5 of k-ras. Telp5 joins the template region of the hTR telomerase subunit. ODNs have been tested in different concentrations (1, 5, 10, 20 micromolar). Cell viability has been tested at 48 and 72 hours. Statistical analysis and graphic design were made with the statistical package "Analyzing Data with GraphPad Prism-1999", GraphPad Sofware Inc., San Diego CA. We used the Student's t test for statistical analysis. RESULTS: The lowest dose (1 microM) was not effective. Using the highest dose (20 microM for 48 hours) of combined AS-KRAS and Telp5 cell viability decreased to 99.67%. The rest of results varied depending on ODN type, dose, and exposure time. CONCLUSIONS: Tested antisense ODNs stop colorectal cancer cell growth, and a combination of anti-telomerase and anti-k-ras is the most useful treatment. Efficacy is best with a higher dose and longer treatment period.

Human alpha 1-antitrypsin gene transfer to in vivo mouse hepatocytes.

The in vivo gene transfer to mouse hepatocytes of pTG 7101, a plasmid containing the full-length gene encoding human alpha 1-antitrypsin (alpha 1-AT) DNA, has been studied by iv administration of recombinant DNA (100 ng/mouse) encapsulated in large and small liposomes. Our results from immunohistochemical liver sections and cytophotometric analysis of hepatocyte chromophore absorbance indicate that human alpha 1-AT was expressed in liver parenchymal cells from mice treated (48 hr before) with DNA encapsulated in small liposomes, and this effect remained for at least 2 weeks. In contrast, the efficiency was greatly limited when large liposomes were used as a vehicle for gene transfer. Additional experiments were performed to study using an ELISA procedure the presence in mouse plasma of human alpha 1-AT from mice treated with encapsulated plasmid in small liposomes or small empty liposomes plus free DNA. According to the immunohistochemical data, the results indicate that detectable alpha 1-AT can only be observed in plasma from mice treated with encapsulated plasmid in small liposomes.

Antitumor effect of B16 melanoma cells genetically modified with the angiogenesis inhibitor rnasin.

The growth of new blood vessels is an essential condition for the development of tumors with a diameter greater than 1-2 mm and also for their metastatic dissemination. RNasin, the placental ribonuclease inhibitor, is known to have antiangiogenic activity through the inhibition of angiogenin and basic fibroblast growth factor. Nevertheless, the administration of the recombinant form of a protein poses several limitations; as a result, we have studied the antitumor effect of RNasin in a murine gene therapy model. RNasin cDNA was subcloned into the pcDNA3 expression vector, and the resulting recombinant plasmid was used to transfect the B16 murine melanoma cell line. An RNasin inverted construction was used as control. Mice intravenously injected with clones expressing RNasin showed a significant inhibition of tumor metastatic progression with respect to control groups (P<.001) and survived longer (P<.001). Tissue sections from RNasin-expressing cell tumors showed a lower number of blood vessels when compared to tissue sections from mice lungs that had been inoculated with control cell lines. The results of these experiments show that the genetic modification of tumor cells with RNasin cDNA yields a significant antitumor effect, and suggest that this effect is at least partially the result of angiogenesis inhibition.

Long-term expression of the human alpha1-antitrypsin gene in mice employing anionic and cationic liposome vector.

The complete process of gene therapy involves three important steps: targeting, delivery, and gene expression. Since each step can be related to the pharmacological concept of affinity, bioavailability, and intrinsic capacity, this commentary examines, from this perspective, the efficiency of anionic and cationic liposomes as vectors for the in vivo gene transfer of the human alpha1-antitrypsin gene. Small liposomes represent the first generation of liposomes destined for the liver parenchymal cell. Although the final efficiency of gene transfer is low, we found that small liposomes are a kind of high-affinity hepatocyte-destined vector because the dose range for mediating the response is three orders of magnitude lower than that used by other procedures. Encapsulated DNA is more efficient than the cationic liposome-DNA complex for in vivo gene transfer. This could be due to gene bioavailability, since encapsulated DNA is protected from enzymatic digestion, whereas DNA externally associated with the liposome can be digested before the complex reaches the target cell. However, when the gene transfer efficiencies of anionic and cationic small liposomes were compared, we observed a similar rate of efficiency and potency, since equivalent plasma levels of human protein were observed after the same i.v. dose of recombinant plasmid encapsulated in anionic or cationic liposomes. On the other hand, the elements selected for constructing the expression cassette greatly influence gene expression and the stability of the gene product, and, therefore, the final efficacy is also limited by the intrinsic capacity of a specific expression cassette to express the gene product.

Long-term expression of the human alpha1-antitrypsin gene in mice employing anionic and cationic liposome vectors.

The efficiency of both anionic and cationic liposomes as vectors for in vivo human alpha1-antitrypsin (AAT) gene transfer was studied in mice with and without an associated partial hepatectomy. The pTG7101 plasmid, containing the full-length human AAT gene, was encapsulated in small liposomes bearing 10% of negatively (phosphatidylserine, PS) or positively (DOTAP) charged lipids. The results indicated that the DNA/lipid ratio was increased in cationic liposomes by inclusion of monosialoganglioside-GM1. The expression of human protein after in vivo gene transfer was quantified in mouse plasma by an ELISA procedure, and revealed that both anionic and cationic liposomes mediated the presence of human protein in mouse plasma for 2-3 weeks. This effect was prolonged (>5 months) when a partial hepatectomy was performed after treatment. In addition, it was observed that the efficacy of liposome-mediated gene transfer was more limited when the plasmid was externally associated to cationic liposomes.

Pharmacodynamic approach to study the gene transfer process employing non-viral vectors.

In the present work we set out to apply pharmacodynamic concepts derived from dose-response curves (Potency and Efficacy) to characterize the gene transfer efficiency of a vector:DNA complex. We employed two widely used vectors, the cationic lipid DOTAP (N,N, N-trimethyl 1-2-3-bis (1-oxo-9-octa-decenyl)oxy-(Z, Z)-1-propanaminium methyl sulfate) and the cationic polymer PEI (polyethylenimine, 800 kDa) to transfect several constructions of the green fluorescent protein cDNA. The analysis of dose-response curves indicated that in all cases the goodness-of-fit was > 0.99. Potency is a measure that provides information on gene activity per amount of DNA. Efficacy is a measure of maximum gene expression achievable using a specific vector:DNA complex, and depends on both the intrinsic efficacy of the gene (evaluated using different vectors to transfer the same gene construct) and on vector efficacy in DNA delivery (evaluated using a single vector to deliver different gene constructs). The results suggest that Potency and Efficacy are objective parameters for describing and comparing the goodness of vectors, as well as the intrinsic efficacy of a given gene construct. Furthermore, they are useful tools that may contribute to a better understanding of the mechanistic gene transfer process of each vector.

Effect of the protein kinase inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine H-7 and N-(2-[methylamino]ethyl)-5-isoquinoline-sulfonamide H-8 on Lewis lung carcinoma tumor progression.

The effects of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine H-7 (a cAMP-dependent protein kinase and protein kinase C inhibitor), n-(2-[methylamino]ethyl)-5-isoquinoline-sulfonamide H-8 (a cAMP- and cGMP-dependent protein kinase inhibitor) and indomethacin (IND, a cyclooxygenase inhibitor) on both the spontaneous metastatic ability of 3LL (Lewis lung carcinoma) tumor cells and anti-tumor host response were studied. The study of tumor progression showed that H-7 and H-8 (2 mg kg(-1) day(-1) , i.p., for 8 days) significantly reduced the mean number of metastases (0.8 +/- 0.2 and 1.0 +/- 0.7, respectively, P < 0.05) with respect to the number of lung metastases (4.2 +/- 2.1) observed in the control group. In turn, the highest tumor-specific cytotoxicity response (50% increase vs. non-treated target cells) was observed when both animal and tumor cells were treated with H-8. This suggests that the protein kinase inhibitors could inhibit tumor progression toward lung metastases formation by blocking the immunosuppressor mechanism triggered by agents that increase intracellular cAMP.

Effects of cimetidine, atropine and prostaglandin E2 on rat mucosal erosions produced by intragastric distension.

The effects of three typical antisecretory agents: cimetidine, atropine and prostaglandin E2 were compared on an acute rat gastric ulcer model which consisted of perfusing the stomach continuously, at a high intraluminal pressure (120 mm H2O), with a simulated gastric juice (0.1 M HCl plus 600 mg pepsin/l). As the acid and pepsin are given exogenously the inhibitory action of the antisecretory drugs is obviated in this model. Cimetidine and atropine failed to reduce gastric erosions, whereas prostaglandin E2 markedly reduced the severity of the mucosal lesions with respect to control values. Long-term treatment with cimetidine also failed to increase the resistance of the gastric mucosa to the digestive action of the artificial gastric juice. These findings indicate that only prostaglandin E2 is cytoprotective and do not support the view that anticholinergics or histamine H2-receptor antagonists have a cytoprotective role on the cells of the gastric mucosa.

Stability of PEI-DNA and DOTAP-DNA complexes: effect of alkaline pH, heparin and serum.

DNA complexes formed with nonviral vectors such as polyethylenimine (PEI) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) are widely used in gene therapy. These complexes prevent the interaction of DNA with the fluorescent probes usually employed to quantify DNA. We thus studied the procedures for DNA quantification from DNA complexes as well as their stability in the presence of DNase or mouse, rat and human sera. Release of the DNA from its complexes was accomplished by increasing the pH of the medium (from 7.3 to 13.4) or by adding heparin. The stability against degradation was tested in vitro, by incubating the complexes at 37 degrees C in the presence of DNase I and sera from the three species. Both high pH and heparin were able to release DNA from its complexes. Naked DNA formed aggregates with serum proteins that delayed electrophoresis migration, and this effect was reversed in the presence of heparin. However, these aggregates did not protect DNA from digestion by serum DNase, and the DNA digesting ability of serum was: mouse>rat>human. The DNA from the complexes was resistant to degradation by DNase I, although a low proportion of DNA from the complexes was partially digested, as determined by electrophoresis. In contrast, PEI-DNA and DOTAP-DNA complexes were stable in the presence of all sera. Heparin and high pH release DNA from its complexes. The order of DNA degradation is: mouse>rat>human, but DOTAP and PEI avoid degradation of DNA by serum compounds.

Antimetastatic effects of liposome entrapped indomethacin.

The "in vivo"administration of sized liposomes encapsulating indomethacin to mice bearing 3LL tumor, significantly reduced the incidence and/or number of superficial lung metastases. Also liposomes encapsulating indomethacin had significant inhibitory effects on the experimentally induced lung metastases. We conclude: i) indomethacin encapsulated in liposomes is more efficient than the free drug in mediating the antimetastatic effects and ii) liposomes are an valuable vehicle in evading the side metastatic effects of this drug during indomethacin treatment of tumor bearing mice.

Morpho-functional study of vascular fluorochrome delivery to lung and liver metastases of Lewis lung carcinoma (3LL).

The growth of 3LL liver and lung metastases related to its vascular organization was studied by morphological and functional methods, using the Hoechst 33342 fluorescent DNA staining technique. Experimental liver and lung metastases were produced in syngeneic C57BL/6 mice by injection of 3LL tumor cells into a lateral tail vein or into the spleen, respectively. The resulting neoplasms were composed of large cells arranged in sheets with a thin irregularly distributed stroma. Scattered blood vessels with an open or closed lumen were observed within the tumor. Functional study of H33342 diffusion showed a single and reticular fluorescent pattern in liver metastases. In contrast, in lung metastases the fluorochrome diffusion revealed two different fluorescent patterns related to the location of the metastasis. Thus, parenchymal and subpleural metastasis presented a fluorescent pattern similar to that observed in the liver whereas metastases located around blood vessels and conducting airways never displayed fluorescence. In summary, our results suggest that the target metastatic organ and/or intra-organ location modulates the characteristics of metabolic exchange of the tumor cells in relation to the vascular organization of the metastatic focus.