Targeting gene therapy for hepatocarcinoma cells with the E. coli purine nucleoside phosphorylase suicide gene system directed by a chimeric alpha-fetoprotein promoter

For hepatocarcinoma (HCC) gene therapy, the tumoricidal efficacy and selective expression of therapeutic gene remain two major challenges. The Escherichia coli (E. coli) purine nucleoside phosphorylase (PNP)/9-(2-deoxy-beta-dribofuranosyl)-6-methylpurine (MeP-dR) suicide gene system exhibits excellent anti-tumor effects, indicating this system directed by a HCC-specific promoter would offer a possibility of targeting gene therapy for HCC. To test this hypothesis, here, we prepared a plasmid (p[HRE]AF/PNP) containing the E. coli PNP/MeP-dR system and a chimeric human alpha-fetoprotein (AFP) promoter, [HRE]AF. We introduced this plasmid into AFP-positive and low-AFP-generating human HCC cells, and evaluated its therapeutic effects on both human HCC cell lines. In the presence of hypoxia, the E. coli PNP gene directed by the [HRE]AF promoter were HCC-specifically expressed in two human HCC cell lines and, moreover, the [HRE]AF-PNP/MeP-dR therapy would yield significant and selective cytotoxicity in both AFP-positive and low-AFP-generating HCC cells. Our findings suggest the [HRE]AF-PNP/MeP-dR therapy has worthy potentialities as an effective strategy for targeting therapy of AFP-positive, and especially AFP-negative or low-AFP-generating HCC.     

RFC方案治疗B细胞幼淋巴细胞白血病一例并文献复习

 【摘要】　目的　探讨B细胞幼淋巴细胞白血病（B-PLL）临床特征。方法　报道1例33岁男性B-PLL患者的临床表现和治疗经过。结果　患者血小板减少、淋巴细胞增高,有全身B症状、伴随脾大和淋巴结肿大。患者拒绝异基因造血干细胞移植,予氟达拉滨、环磷酰胺联合利妥昔单抗组成的RFC方案化疗后,患者达到血液、骨髓、免疫表型完全缓解。结论　RFC方案可能是B-PLL合适且有效的初始治疗选择之一。     

The effect of arylazido aminopropionyl ATP (ANAPP3) on inhibition of pelvic nerve evoked contractions of the cat urinary bladder

Previously reported work has provided evidence that arylazido aminopropionyl ATP (ANAPP₃), a P₂-receptor antagonist, blocks the effects of ATP and other purine analogs, in the urinary bladder of the cat. This antagonism appeared limited to those effects mediated by P₂-receptors, such as contraction of the urinary bladder. However, it was noted that ANAPP₃ did alter the effects of adenosine, mediated through P₁-receptors, in some preliminary experiments. A series of experiments was undertaken to determine if ANAPP₃ blocks the P₁-receptors and/or has effects on inhibition of bladder contractions. The urinary tract of anesthetized cats was exposed by a midline abdominal incision. The pelvic and hypogastric nerves were isolated and prepared for electrical stimulation. Bladder contractions were induced by stimulation of the pelvic nerves every 30s with square wave pulses. These contractions were inhibited by hypogastric nerves and the exogenous administration of ATP, noradrenaline, adenosine or β, γ-methylene ATP (APPCP), a hydrolysis resistant analog of ATP. ANAPP₃ (0.5 μmol, i.a.) was administered, but it did not antagonize the inhibition induced by any of the exogenously administered agents or hypogastric nerve stimulation. This study indicates that ANAPP₃ is specific for P₂-receptors and is not an effective antagonist of P₁-receptors, at least in the urinary tract of the cat.     

Rekomendacje diagnostyczne i terapeutyczne dla przewlekłej białaczki limfocytowej w 2014 r. – raport Grupy Roboczej PTHiT oraz PALG – CLL

Chronic lymphocytic leukemia (CLL) is predominantly a disease of the elderly, with a median age of 70 years at diagnosis. Appropriate treatment approach requires physical examination, evaluation of performance status, co-morbidities and severity of comorbid conditions, biological age, peripheral, blood count with morphologic examination. In addition, in some patients bone marrow evaluation, serum biochemistry including serum lactate dehydrogenasis and β₂-microglobulin, Coombs’ test, imaging of adenomegalies, assessed either by CT scan or by the combination of chest X-ray and abdomen ultrasound should be incorporated into decision-making process with regard to intensity of treatment. In patients requiring therapy, particularly a second-line treatment, del 17p and/or p53 mutations should be also checked. Elderly and/or comorbid patients with CLL may not tolerate more aggressive approach due to high risk of unacceptable toxicity of purine nucleoside analogs, especially in combination with cyclophosphamide and rituximab. Therefore in these patients population, chlorambucil +/- rituximab is still accepted as the first-line treatment and chlorambucil remains the backbone of treatment against which the new protocols should be tested. The results of ongoing trials investigating the role of new immunochemotherapies and the other novel agents will probably modify the treatment of CLL in the following years. In this article we present recommendations for diagnosis, prognosis and therapy of CLL elaborated by by PTH i T and PALG-CLL group.     

High Risk Screening of Adenosine Deaminase and Purine Nucleoside Phosphorylase Deficiency in Japan

Seventy-three patients with immunodeficiency syndromes or frequent infections were screened for adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency by the dried blood spot paper method. Among 23 patients with combined immunodeficiency, five patients with ADA deficiency were detected. In the light of the incidence of primary immunodeficiencies and financial limitation, it seems to be adequate to put screening for ADA & PNP deficiency in force for all infants with signs of an immune defect.     

分光光度法测定红细胞嘌呤核苷磷酸化酶

应用Trinder反应建立终点分光光度法测定红细胞嘌呤核苷磷酸化酶(PNP)活性,操作简单、快速、灵敏、准确。对酶反应最佳条件进行了实验探讨,底物Km为64mm,最佳PH7.5,延滞期1分钟30秒,1.5～8分钟酶活性与反应时间成比例。用3.5mol/L硫酸铵溶液终止反应,效果好,显色稳定。批内CV2.51％～3.15％,批间CV2.69％～3.28％。35例正常人红细胞PNP活性为13.66±1.73u/ml红细胞。     

Blockade by 1,3-dipropyl-8-cyclopentylxanthine (CPX) of purine protection against kainate neurotoxicity

The adenosine A₁ receptor selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (CPX) has been administered systemically to rats together with the neurotoxin kainic acid. At the lower doses of CPX tested, 10 and 50 μg/kg, which were sufficient to prevent the neuroprotective activity of exogenous agonists, there was no exacerbation of the neuronal damage. At 250 μg/kg, some enhancement of damage was found, which was also produced by 8-(p-sulphopenyl)theophylline, a non-selective xanthine which does not cross the blood-brain barrier. The results are consistent with the involvement of a central A₁ receptor in the neuroprotective activity of purines, and suggest that blockade of a peripheral adenosine receptor, possibly of the A₂ type, may increase neuronal damage.     

Effects of two nucleoside transport inhibitors, dipyridamole and soluflazine, on purine release from the rat cerebral cortex

The effects of two nucleoside transport inhibitors, dipyridamole and soluflazine, on adenosine, inosine and oxypurine release from the normoxic and hypoxic/ischemic rat cerebral cortex have been studied. Dipyridamole (500 μg/kg) enhanced adenosine release during hypoxic/ischemic challenges in comparison with saline-injected controls. It decreased the hypoxia/ischemia-elicited releases of inosine, hypoxanthine and xanthine. Both basal and hypoxia/ischemia-elicited releases of uric acid were elevated. Soluflazine, administered topically or systemically, failed to enhance adenosine release and did not consistently alter the hypoxia/ischemia-evoked releases of inosine, hypoxanthine and xanthine. Basal release of uric acid was elevated. The failure of either drug to elevate the basal or hypoxia/ischemia-evoked releases of adenosine above predrug levels illustrates one of the problems which may be inherent in the use of bidirectional nucleoside transport inhibitors for the manipulation of adenosine levels in the cerebral interstitial fluid.     

Glucose deprivation increases basal and electrically evoked transmitter release from rat striatal slices. Role of NMDA and adenosine A1 receptors

We have investigated how glucose deprivation in vitro influences the basal and electrically evoked release of dopamine and acetylcholine from rat striatal slices and the role of endogenous activation of NMDA receptors and adenosine A₁ receptors in determining the magnitude of this response. Rat striatal slices, preincubated with [

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]dopamine and [

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]choline, were superfused continuously and stimulated electrically. Before and during the second stimulation, some slices were superfused with glucose-free Krebs' solution. Such glucose deprivation caused a 2 to 3-fold increase of the electrically evoked, calcium-dependent release of endogenous adenosine (but not hypoxanthine and inosine) and [

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]dopamine and a 30% increase in release of [

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]acetylcholine. Glucose deprivation also caused a delayed increase in the release of [

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]dopamine, but not of [

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]acetylcholine. The dopamine release was not calcium dependent. The addition of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 μM), a selective adenosine A₁ receptor antagonist, slightly enhanced the glucose deprivation-induced stimulatory effect on the evoked release of these two transmitters, whereas the NMDA receptor antagonist dizocilpine((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine; 3 μM) markedly attenuated the stimulatory effect of glucose deprivation. The change in basal dopamine release was not influenced by DPCPX, but was slightly attenuated by dizocilpine. In summary, the results suggest that lack of substrate induces release of both glutamate, which by actions on presynaptic NMDA receptors causes the release of dopamine, and of adenosine, which via adenosine A₁ receptors reduces the electrically evoked release of both dopamine and acetylcholine.     

Synthesis and biological activity of 8-azapurine and pyrazolo[4,3-d]pyrimidine analogues of myoseverin

The trisubstituted purine myoseverin has been recently identified as a novel inhibitor of microtubule assembly. To analyze the effects of modifying its heterocyclic skeleton, we prepared 8-azapurine and pyrazolo[4,3-d]pyrimidine analogues of myoseverin and compared their biological activities. Rearrangement of nitrogen atoms in the heterocycle changes the affinity of the compounds to purified tubulin, as demonstrated by the results of polymerization assays, and affects the proliferation of cancer cell lines. Surprisingly, compound E2GG, a pyrazolo[4,3-d]pyrimidine analogue of myoseverin, displayed inhibitory activity towards both tubulin polymerization and the activity of cyclin-dependent kinases 1, 2 and 7. Such a dual specificity-inhibitor offers a starting point for developing a novel class of antiproliferative agents.