Cyclooxygenase-2-linked attenuation of hypoxia-induced pulmonary hypertension and intravascular thrombosis

Exogenous prostacyclin is effective in reducing pulmonary vascular resistance in some forms of human pulmonary hypertension (PH). To explore whether endogenous prostaglandins played a similar role in pulmonary hypertension, we examined the effect of deleting cyclooxygenase (COX)-gene isoforms in a chronic hypoxia model of PH. Pulmonary hypertension, examined by direct measurement of right ventricular end systolic pressure (RVESP), right ventricular hypertrophy (n = 8), and hematocrit (n = 3), was induced by 3 weeks of hypobarichypoxia in wild-type and COX-knockout (KO) mice. RVESP was increased in wild-type hypoxic mice compared with normoxic controls (24.4 ± 1.4 versus 13.8 ± 1.9 mm Hg; n = 8; p < 0.05). COX-2 KO mice showed a greater increase in RVESP following hypoxia (36.8 ± 2.7 mm Hg; p < 0.05). Urinary thromboxane (TX)B2 excretion increased following hypoxia (44.6 ± 11.1 versus 14.7 ± 1.8 ng/ml; n = 6; p < 0.05), an effect that was exacerbated by COX-2 gene disruption (54.5 ± 10.8 ng/ml; n = 6). In contrast, the increase in 6-keto-prostacyclin1α excretion following hypoxia was reduced by COX-2 gene disruption (29 ± 3 versus 52 ± 4.6 ng/ml; p < 0.01). Tail cut bleed times were lower following hypoxia, and there was evidence of intravascular thrombosis in lung vessels that was exacerbated by disruption of COX-2 and reduced by deletion of COX-1. The TXA2/endoperoxide receptor antagonist ifetroban (50 mg/kg/day) offset the effect of deleting the COX-2 gene, attenuating the hypoxia-induced rise in RVESP and intravascular thrombosis. COX-2 gene deletion exacerbates pulmonary hypertension, enhances sensitivity to TXA2, and induces intravascular thrombosis in response to hypoxia. The data provide evidence that endogenous prostaglandins modulate the pulmonary response to hypoxia. Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics.

Inheritance of qualitative and quantitative trypsin inhibitor variants in Pisum

A trypsin inhibitor locus (Tri) has been mapped close to Vc-2 on Pisum (pea) linkage group 5 using recombinant inbred lines derived from crosses of genotypes showing qualitative variation in seed trypsin inhibitors. F2 seed populations derived from crosses between lines showing qualitative variation in trypsin inhibitors as well as quantitative variation in inhibitor activity showed an association between the segregation of the structural variation and relative activity levels. Clones complementary to Pisum trypsin inhibitor mRNA were used in hybridization analyses which showed that the segregation of protein polymorphisms reflected directly the segregation of polymorphisms associated with the structural genes.

UNIFOLIATA regulates leaf and flower morphogenesis in pea

Background The vegetative phenotype of the pea mutant unifoliata (uni) is a simplification of the wild-type compound leaf to a single leaflet. Mutant uni plants are also self-sterile and the flowers resemble known floral meristem and organ identity mutants. In Antirrhinum and Arabidopsis, mutations in the floral meristem identity gene FLORICAULA/LEAFY (FLO/LFY) affect flower development alone, whereas the tobacco FLO/LFY homologue, NFL, is expressed in vegetative tissues, suggesting that NFL specifies determinacy in the progenitor cells for both flowers and leaves. In this paper, we characterised the pea homologue of FLO/LFY. Results The pea cDNA homologue of FLO/LFY, PEAFLO, mapped to the uni locus in recombinant-inbred mapping populations and markers based on PEAFLO cosegregated with uni in segregating sibling populations. The characterisation of two spontaneous uni mutant alleles, one containing a deletion and the other a point mutation in the PEAFLO coding sequences, predicted that PEAFLO corresponds to UNI and that the mutant vegetative phenotype was conferred by the defective PEAFLO gene. Conclusions The uni mutant demonstrates that there are shared regulatory processes in the morphogenesis of leaves and flowers and that floral meristem identity genes have an extended role in plant development. Pleiotropic regulatory genes such as UNI support the hypothesis that leaves and flowers derive from a common ancestral sporophyll-like structure. The regulation of indeterminacy during leaf and flower morphogenesis by UNI may reflect a primitive function for the gene in the pre-angiosperm era.

Irradiation, cisplatin, and 5-azacytidine upregulate cytomegalovirus promoter in tumors and muscles: Implementation of non-invasive fluorescence imaging

Purpose: The cytomegalovirus (CMV) promoter is one of the most commonly used promoters for expression of transgenes in mammalian cells. The aim of our study was to evaluate the role of methylation and upregulation of the CMV promoter by irradiation and the chemotherapeutic agent cisplatin in vivo using non-invasive fluorescence in vivo imaging. Procedures: Murine fibrosarcoma LPB and mammary carcinoma TS/A cells were stably transfected with plasmids encoding CMV and p21 promoter-driven green fluorescent protein (GFP) gene. Solid TS/A tumors were induced by subcutaneous injection of fluorescent tumor cells, while leg muscles were transiently transfected with plasmid encoding GFP under the control of the CMV promoter. Cells, tumors, and legs were treated either by DNA methylation inhibitor 5-azacytidine, irradiation, or cisplatin. GFP expression was determined using a fluorescence microplate reader in vitro and by non-invasive fluorescence imaging in vivo. Results: Treatment of cells, tumors, and legs with 5-azacytidine (re)activated the CMV promoter. Furthermore, treatment with irradiation or cisplatin resulted in significant upregulation of GFP expression both in vitro and in vivo. Conclusions: Observed alterations in the activity of the CMV promoter limit the usefulness of this widely used promoter as a constitutive promoter. On the other hand, inducibility of CMV promoters can be beneficially used in gene therapy when combined with standard cancer treatment, such as radiotherapy and chemotherapy. © 2010 The Author(s).

Radiosensitizing effect of electrochemotherapy in a fractionated radiation regimen in radiosensitive murine sarcoma and radioresistant adenocarcinoma tumor model

Radiosensitizing Effect of Electrochemotherapy in a Fractionated Radiation Regimen in Radiosensitive Murine Sarcoma and Radioresistant Adenocarcinoma Tumor Model. Electrochemotherapy can potentiate the radiosensitizing effect of bleomycin, as shown in our previous studies. To bring this treatment closer to use in clinical practice, we evaluated the interaction between electrochemotherapy with bleomycin and single-dose or fractionated radiation in two murine tumor models with different histology and radiosensitivity. Radiosensitive sarcoma SA-1 and radioresistant adenocarcinoma CaNT subcutaneous tumors grown in A/J and CBA mice, respectively, were used. The anti-tumor effect and skin damage around the treated tumors were evaluated after electrochemotherapy with bleomycin alone or combined with single-dose radiation or a fractionated radiation regimen. The anti-tumor effectiveness of electrochemotherapy was more pronounced in SA-1 than CaNT tumors. In both tumor models, the tumor response to radiation was not significantly influenced by bleomycin alone or by electroporation alone. However, electrochemotherapy before the first tumor irradiation potentiated the response to a single-dose or fractionated radiation regimen in both tumors. For the fractionated radiation regimen, normal skin around the treated tumors was damaged fourfold less than for the single-dose regimen. Electrochemotherapy prior to single-dose irradiation induced more damage to the skin around the treated tumors and greater loss of body weight compared to other irradiated groups, whereas electrochemotherapy combined with the fractionated radiation regimen did not. Electrochemotherapy with low doses of bleomycin can also be used safely for radiosensitization of different types of tumors in a fractionated radiation regimen, resulting in a good anti-tumor effect and no major potentiating effect on radiation-induced skin damage. © 2009 by Radiation Research Society.

The effect of the histological properties of tumors on transfection efficiency of electrically assisted gene delivery to solid tumors in mice

Uniform DNA distribution in tumors is a prerequisite step for high transfection efficiency in solid tumors. To improve the transfection efficiency of electrically assisted gene delivery to solid tumors in vivo, we explored how tumor histological properties affected transfection efficiency. In four different tumor types (B16F1, EAT, SA-1 and LPB), proteoglycan and collagen content was morphometrically analyzed, and cell size and cell density were determined in paraffin-embedded tumor sections under a transmission microscope. To demonstrate the influence of the histological properties of solid tumors on electrically assisted gene delivery, the correlation between histological properties and transfection efficiency with regard to the time interval between DNA injection and electroporation was determined. Our data demonstrate that soft tumors with larger spherical cells, low proteoglycan and collagen content, and low cell density are more effectively transfected (B16F1 and EAT) than rigid tumors with high proteoglycan and collagen content, small spindle-shaped cells and high cell density (LPB and SA-1). Furthermore, an optimal time interval for increased transfection exists only in soft tumors, this being in the range of 5-15 min. Therefore, knowledge about the histology of tumors is important in planning electrogene therapy with respect to the time interval between DNA injection and electroporation.

Controlled systemic release of interleukin-12 after gene electrotransfer to muscle for cancer gene therapy alone or in combination with ionizing radiation in murine sarcomas

Background: The present study aimed to evaluate the antitumor effectiveness of systemic interleukin (IL)-12 gene therapy in murine sarcoma models, and to evaluate its interaction with the irradiation of tumors and metastases. To avoid toxic side-effects of IL-12 gene therapy, the objective was to achieve the controlled release of IL-12 after intramuscular gene electrotransfer. Methods: Gene electrotransfer of the plasmid pORF-mIL12 was performed into the tibialis cranialis in A/J and C57BL/6 mice. Systemic release of the IL-12 was monitored in the serum of mice after carrying out two sets of intramuscular IL-12 gene electrotransfer of two different doses of plasmid DNA. The antitumor effectiveness of IL-12 gene electrotransfer alone or in combination with local tumor or lung irradiation with X-rays, was evaluated on subcutaneous SA-1 and LPB tumors, as well as on lung metastases. Results: A synergistic antitumor effect of intramuscular gene electrotransfer combined with local tumor irradiation was observed as a result of the systemic distribution of IL-12. The gene electrotransfer resulted in up to 28% of complete responses of tumors. In combination with local tumor irradiation, the curability was increased by up to 100%. The same effect was observed for lung metastases, where a potentiating factor of 1.3-fold was determined. The amount of circulating IL-12 was controlled by the number of repeats of gene electrotransfer and by the amount of the injected plasmid. Conclusions: The present study demonstrates the feasibility of treatment by IL-12 gene electrotransfer combined with local tumor or lung metastases irradiation on sarcoma tumors for translation into the clinical setting. Copyright © 2009 John Wiley & Sons, Ltd.

Gene electrotransfer into murine skeletal muscle: A systematic analysis of parameters for long-term gene expression

Skeletal muscle is an attractive target tissue for delivery of therapeutic genes, since it is well vascularized, easily accessible, and has a high capacity for protein synthesis. For efficient transfection in skeletal muscle, several protocols have been described, including delivery of low voltage electric pulses and a combination of high and low voltage electric pulses. The aim of this study was to determine the influence of different parameters of electrotransfection on short-term and long-term transfection efficiency in murine skeletal muscle, and to evaluate histological changes in the treated tissue. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses, and different doses of plasmid DNA were tested for electrotransfection of tibialis cranialis muscle of C57BI/6 mice using DNA plasmid encoding green fluorescent protein (GFP). Transfection efficiency was assessed on frozen tissue sections one week after electrotransfection using a fluorescence microscope and also noninvasively, followed by an in vivo imaging system using a fluorescence stereo microscope over a period of several months. Histological changes in muscle were evaluated immediately or several months after electrotransfection by determining infiltration of inflammatory mononuclear cells and presence of necrotic muscle fibers. The most efficient electrotransfection into skeletal muscle of C57BI/6 mice in our experiments was achieved when one high voltage (HV) and four low voltage (LV) electric pulses were applied 5 seconds after the injection of 30 μg of plasmid DNA. This protocol resulted in the highest short-term as well as long-term transfection. The fluorescence intensity of the transfected area declined after 2-3 weeks, but GFP fluorescence was still detectable 18 months after electrotransfection. Extensive inflammatory mononuclear cell infiltration was observed immediately after the electrotransfection procedure using the described parameters, but no necrosis or late tissue damage was observed. This study showed that electric pulse parameters, time lag between the injection of DNA and application of electric pulses, and dose of plasmid DNA affected the duration of transgene expression in murine skeletal muscle. Therefore, transgene expression in muscle can be controlled by appropriate selection of electrotransfection protocol.

Myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones include double-positive CD8+CD4+ T cells : evidence from myeloma-bearing mouse model

The graft-versus-myeloma (GVM) effect represents a powerful form of immune attack exerted by alloreactive T cells against multiple myeloma cells, which leads to clinical responses in multiple myeloma transplant recipients. Whether myeloma cells are themselves able to induce alloreactive T cells capable of the GVM effect is not defined. Using adoptive transfer of T naive cells into myeloma-bearing mice (established by transplantation of human RPMI8226-TGL myeloma cells into CD122(+) cell-depleted NOD/SCID hosts), we found that myeloma cells induced alloreactive T cells that suppressed myeloma growth and prolonged survival of T cell recipients. Myeloma-induced alloreactive T cells arising in the myeloma-infiltrated bones exerted cytotoxic activity against resident myeloma cells, but limited activity against control myeloma cells obtained from myeloma-bearing mice that did not receive T naive cells. These myeloma-induced alloreactive T cells were derived through multiple CD8(+) T cell divisions and enriched in double-positive (DP) T cells coexpressing the CD8alphaalpha and CD4 coreceptors. MHC class I expression on myeloma cells and contact with T cells were required for CD8(+) T cell divisions and DP-T cell development. DP-T cells present in myeloma-infiltrated bones contained a higher proportion of cells expressing cytotoxic mediators IFN-gamma and/or perforin compared with single-positive CD8(+) T cells, acquired the capacity to degranulate as measured by CD107 expression, and contributed to an elevated perforin level seen in the myeloma-infiltrated bones. These observations suggest that myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones are enriched with DP-T cells equipped with cytotoxic effector functions that are likely to be involved in the GVM effect.

Enhancement of the chemoprotective enzymes glucuronosyl transferase and glutathione transferase in specific organs of the rat by the coffee components kahweol and cafestol

The coffee components kahweol and cafestol (K/C) have been reported to protect the colon and other organs of the rat against the formation of DNA adducts by 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) and aflatoxin B1. PhIP is a cooked-food mutagen to which significant human exposure and a role in colon cancer etiology are attributed, and, interestingly, such cancers appear to develop at a lower rate in consumers of coffees with high amounts of K/C. Earlier studies in rodent liver have shown that a key role in the chemopreventive effect of K/C is likely to be due to the potential of these compounds to induce the detoxification of xenobiotics by glutathione transferase (GST) and to enhance the synthesis of the corresponding co-factor glutathione. However, mutagens like PhIP may also be detoxified by UDP-glucuronosyl transferase (UDPGT) for which data are lacking regarding a potential effect of K/C. Therefore, in the present study, we investigated the effect of K/C on UDPGT and, concomitantly, we studied overall GST and the pattern of individual GST classes, particularly GST-θ, which was not included in earlier experiments. In addition, we analyzed the organ-dependence of these potentially chemopreventive effects. K/C was fed to male F344 rats at 0.122% in the chow for 10 days. Enzyme activities in liver, kidney, lung, colon, salivary gland, pancreas, testis, heart and spleen were quantified using five characteristic substrates and the hepatic protein pattern of GST classes α, μ, and π was studied with affnity chromatography/HPLC. Our study showed that K/C is not only capable of increasing overall GST and GST classes α, μ, and π but also of enhancing UDGPT and GST-θ. All investigated K/C effects were strongest in liver and kidney, and some response was seen in lung and colon but none in the other organs. In summary, our results show that K/C treatment leads to a wide spectrum of increases in phase II detoxification enzymes. Notably, these effects occurred preferentially in the well perfused organs liver and kidney, which may thus not only contribute to local protection but also to anti-carcinogenesis in distant, less stimulated organs such as the colon.

Influence of cytochrome P-450 inhibitors on the inhalative uptake of methyl chloride and methylene chloride in male B6C3F1 mice

Dichloromethane (DCM) is thought to be metabolized in vivo by two independent pathways: a glutathione (GSH) dependent pathway that yields CO2 and a cytochrome P-450 mediated one that yields both CO and CO2 (Gargas et al 1986). With a physiologically based pharmacokinetic (PB-PK) model, Andersen et al (1987) calculate the quantitative parameters for both metabolic pathways. Using the kinetic parameters thus obtained and the results of two carcinogenicity studies with rodents (Serota et al 1986; NTP 1985), the authors then estimate the tumour risk for humans.

Identification of theta-class glutathione S-transferase in liver cytosol of the marmoset monkey

The presence of theta-class glutathione S-transferase (GST) in marmoset monkey liver cytosol was investigated. An anti-peptide antibody targeted against the C-terminus of rGSTT1 reacted with a single band in marmoset liver cytosol that corresponded to a molecular weight of 28 kDa. The intensity of the immunoreactive band was not affected by treatment of marmoset monkeys with 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbitone, rifampicin or clofibric acid. Similarly, activity towards methyl chloride (MC) was unaffected by these treatments. However, GST activity towards 1,2-epoxy3-(p- nitrophenoxy)-propane (EPNP) was increased in marmosets treated with phenobarbitone (2.6-fold) and rifampicin (2.6-fold), activity towards dichloromethane (DCM) was increased by 50% after treatment of marmosets with clofibric acid, and activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was raised slightly (30-42% increases) after treatment with phenobarbitone, rifampicin or clofibric acid. Compared with humans, marmoset liver cytosol GST activity towards DCM was 18-fold higher, activity towards MC was 7 times higher and activity towards CDNB was 4 times higher. Further, EPNP activity was clearly detectable in marmoset liver cytosol samples, but was undetectable in human samples. Immunoreactive marmoset GST was partially purified by affinity chromatography using hexylglutathione-Sepharose and Orange A resin. The interaction of immunoreactive marmoset GST was similar to that found previously for rat and human GSTT1, suggesting that this protein is also a theta class GST. However, unlike rat GSTT1, the marmoset enzyme was not the major catalyst of EPNP conjugation. Instead, immunoreactivity was closely associated with activity towards MC. In conclusion, these results provide evidence for the presence of theta-class GST in the marmoset monkey orthologous to rGSTT1 and hGSTT1.

Determination of glutathione transferase (GSTT1-1) activities in different tissues based on formation of radioactive metabolites using 35S-glutathione

A new system has been developed to determine enzyme activities of glutathione transferase θ (GSTT1-1) based on radiometric product detection resulting from the enzymic reaction of methyl chloride with 35S-labelled glutathione. In principle, the method is universally applicable for determination of glutathione transferase activities towards a multiplicity of substrates. The method distinguishes between erythrocyte GSTT1-1 activities of human 'non-conjugators', 'low conjugators' and 'high conjugators'. Application to cytosol preparations of livers and kidneys of male and female Fischer 344 and B6C3F1 mice reveals differential GSTT1-1 activities in hepatic and renal tissues. These ought to be considered in species-specific modellings of organ toxicities of chlorinated hydrocarbons.

DNA binding study of isophorone in rats and mice

Following isophorone exposure, in a 2-year study with F344 rats and B6C3F1 mice performed under the National Toxicology Program (NTP), an elevated incidence of tumors was observed in male rats (kidney tumors) and male mice (liver tumors). Female rats and mice showed no elevation of tumor rates by isophorone (NTP 1986).

Obesity and hypertension have differing oxidant handling molecular pathways in age-related chronic kidney disease

Chronic kidney disease (CKD) in ageing is a burden on health systems worldwide. Rat models of age-related CKD linked with obesity and hypertension were used to investigate alterations in oxidant handling and energy metabolism to identify gene targets or markers for age-related CKD. Young adult (3 months) and old (21–24 months) spontaneously-hypertensive (SHR), normotensive Wistar-Kyoto (WKY) and Wistar rats (normotensive, obese in ageing) were compared for renal functional and physiological parameters, renal fibrosis and inflammation, oxidative stress (hemeoxygenase-1/HO-1), apoptosis and cell injury (including Bax:Bcl-2), phosphorylated and non-phosphorylated forms of oxidant and energy sensing proteins (p66Shc, AMPK), signal transduction proteins (ERK1/2, PKB), and transcription factors (NF-κB, FoxO1). All old rats were normoglycemic. Renal fibrosis, tubular epithelial apoptosis, interstitial macrophages and myofibroblasts (all p < 0.05), p66Shc/phospho-p66 (p < 0.05), Bax/Bcl-2 ratio (p < 0.05) and NF-κB expression (p < 0.01) were highest in old obese Wistars. Expression of phospho-FoxO/FoxO was elevated in old Wistars (p < 0.001) and WKYs (p < 0.01). SHRs had high levels in young and old rats. Expression of PKB, phospho-PKB, ERK1/2 and phospho-ERK1/2 were significantly elevated in all aged animals. These results suggest that obesity and hypertension have differing oxidant handling and signalling pathways that act in the pathogenesis of age-related CKD