Alterations in [3H]thymidine incorporation into DNA and [3H]uridine incorporation into RNA induced by 5-azacytidine in vivo.

Administration in vivo of 5-azacytidine (5-aza-CR) caused suppression of [³H]thymidine ([³H]TdR) incorporation into DNA of bone marrow and gastrointestinal mucosa of mice and a more prolonged suppression of L1210 ascites tumor. Single doses of 5-aza-CR caused a modest and short-lived suppression of incorporation of [³H]uridine ([³H]UR) into nuclear RNA of L1210 ascites tumor cells. No suppression of [³H]UR incorporation into RNA of bone marrow or gastrointestinal mucosa was observed. L1210 tumor cells resistant to the other active cytidine analogue, cytosine arabinoside, demonstrated less disruption of [³H]TdR incorporation after exposure to 5-aza-CR, suggesting some cross resistance in the effects of these two drugs on DNA synthesis. Survival studies carried out in mice bearing both the sensitive and resistant L1210 tumor cell lines confirmed cross resistance of the anti-tumor effects of the two cytidine analogues. Second doses of 5-aza-CR, with the timing og administration based upon the differing patterns of recovery of [³H]TdR incorporation between normal tissues and tumor cells, led to a prolongation of survival in mice bearing the sensitive L1210 ascites tumor.     

Arabinose furanosyl thymidine: uptake, phosphorylation and incorporation into DNA of mammalian cells

The naturally occurring nucleoside analogue arabinosyl thymidine is known as an anti-herpes and anti-cancer agent. The biologically active form is arabinosyl thymidine triphosphate (Ara-TTP), which inhibits cellular and viral DNA-polymerases and thus interferes with DNA replication. Using two murine erythroleukemia cell lines, Friend cell clone F4-6 and F4-12N, the latter being thymidine kinase deficient (TK-) cells transformed to a TK+ phenotype with the HSV TK gene, we have determined 1) the role of cellular and herpes simplex virus thymidine kinase (HSV TK) in the uptake of Ara-T into the cells; 2) the subsequent phosphorylation of intracellular Ara-T to Ara-TMP, Ara-TDP and Ara-TTP; 3) the incorporation of Ara-TTP into the DNA. Incorporation into DNA was studied under different conditions, including selective inhibition of the different cellular DNA polymerases by aphidicolin (that inhibits polymerases alpha and delta) and dideoxythymidine (that preferentially inhibits polymerases beta and gamma). The uptake of Ara-T into the methanol soluble pool of the cells depends upon its phosphorylation to Ara-TMP, which is more efficiently performed by the HSV TK than by the cellular TK, thus explaining the sensitivity of HSV infected cells to Ara-T. However, using increasing concentrations of Ara-T, we have shown that phosphorylation also occurs in normal control cells due to the cellular thymidine kinase. More than 90% of Ara-T is phosphorylated in the cell, and more than 60% of total Ara-T(MP, DP, TP) exists in the triphosphate form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Loss of murine tumor thymidine kinase activity in vivo following 5-fluorouracil (FUra) treatment by incorporation of FUra into RNA.

The effects of 5-fluorouracil (FUra) treatment on thymidine kinase (TKase) activity were examined in vivo in CD8F1 mice bearing first generation CD8F1 mouse mammary tumors. TKase activity was not affected by low dose FUra25 (25 mg/kg), a dose which substantially inhibited thymidylate synthase (TSase), but was severely inhibited 24 hr following treatment with FUra100, a weekly maximally tolerated dose, as judged by activity measurements and labeling of DNA with [3H]thymidine. The amount of (FU)RNA was increased markedly with increasing FUra dose from 0.4 nmol/mg DNA at FUra25 to 2.2 nmol/mg DNA at FUra100. At FUra100, TKase activity gradually declined over 24 hr to less than 10% of the control value, remained low for a further 48 hr, and then was gradually restored to control levels by 168 hr. The loss of TKase activity followed the incorporation of FUra into RNA which peaked at 4-5 hr. TKase activity was not restored by removal of endogenous inhibitors but was restored by treatment with uridine. TKase activity was not inhibited by therapeutic levels of methotrexate (300 mg/kg). TKase from murine colon 38 carcinoma was also severely inhibited, but the activity from colon 26 was only partially (50%) inhibited. Ornithine decarboxylase was also inhibited by FUra100 treatment in the CD8F1 tumor. These results demonstrate that certain short-lived, proliferation-related enzymes are affected by FUra doses higher than those required for TSase inhibition, and this effect appears to correlate with incorporation of FUra into RNA. Thus, in some tumors high doses of FUra can inhibit salvage as well as de novo synthesis of thymidylate providing an increased block of DNA synthesis and increased therapeutic advantage.     

强心苷类化合物诱导肿瘤细胞自噬的研究进展

强心苷类化合物(cardiac glycosides,CGs)作为钠泵调节药物一直在临床上用于治疗心脏疾病,但近年来其抗肿瘤的作用逐渐引起关注。大量研究显示CGs可通过诱导细胞自噬发挥抗肿瘤效应,介导的信号传导通路包括MAPK、PI3K-Akt-m TOR等。本文就CGs诱导肿瘤细胞自噬作用的研究现状和进展作一综述。     

Activation of macrophages and lymphocytes by methylglyoxal against tumor cells in the host

Methylglyoxal is a normal metabolite and has the potential to affect a wide variety of cellular processes. In particular, it can act selectively against malignant cells. The study described herein was to investigate whether methylglyoxal can enhance the non-specific immunity of the host against tumor cells. Methylglyoxal increased the number of macrophages in the peritoneal cavity of both normal and tumor-bearing mice. It also elevated the phagocytic capacity of macrophages in both these groups of animals. This activation of macrophages was brought about by increased production of Reactive Oxygen Intermediates (ROIs) and Reactive Nitrogen Intermediates (RNIs). The possible mechanism for the production of ROIs and RNIs can be attributed to stimulation of the respiratory burst enzyme NADPH oxidase and iNOS, respectively. IFN-gamma, which is a regulatory molecule of iNOS pathway also showed an elevated level by methylglyoxal. TNF-alpha, which is an important cytokine for oxygen independent killing by macrophage also increased by methylglyoxal in both tumor-bearing and non tumor-bearing animals. Methylglyoxal also played a role in the proliferation and cytotoxicity of splenic lymphocytes. In short, it can be concluded that methylglyoxal profoundly stimulates the immune system against tumor cells.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on [3H]thymidine incorporation into rat liver deoxyribonucleic acid

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on [3H]thymidine incorporation into hepatic DNA was studied in rats. In non-hepatectomized male and female animals, incorporation measured at the peak of the first round of liver DNA synthesis after TCDD treatment (10 micrograms/kg) was similar to that of control animals. In contrast, the first round of [3H]thymidine incorporation after a 1/3 hepatectomy was enhanced 3-fold in TCDD-treated rats. The enhanced response to 1/3 hepatectomy was produced by doses of TCDD ranging from 1 to 30 micrograms/kg with an apparent ED50 of 5 micrograms/kg. Enhanced incorporation was observed when the 1/3 hepatectomy was performed 5-10 days after an ED50 dose and it returned to the control level after 20 days. This enhanced response was not preceded by changes in food consumption or hepatic activities of ornithine decarboxylase (ODC), tyrosine aminotransferase (TAT) or gamma glutamyl transpeptidase (GGT) when compared to respective control values. Also, the enhanced incorporation was not necessarily due to removal of 1/3 of the liver because it was also seen in TCDD-treated rats that were laparotomized. The mechanism of enhancement in laparotomized animals does not appear to involve a diminished response of the liver to the inhibitory effects of adrenal hormones on liver DNA synthesis. This was suggested by the finding that an adrenalectomy prior to the laparotomy did not block the enhanced incorporation of [3H]thymidine into hepatic DNA. The mechanism by which TCDD enhances the first round of liver DNA synthesis after a 1/3 hepatectomy or laparotomy remains to be determined.     

青蒿琥酯诱导肿瘤细胞凋亡与抑制存活蛋白表达有关

目的 :探讨青蒿琥酯 (artesunate ,Art)诱导肿瘤细胞凋亡与存活蛋白 (survivinprotein)表达的关系。方法 :采用细胞荧光染色、流式细胞术、琼脂糖凝胶电泳法和测定细胞浆Caspase 3的活性等手段检测肿瘤细胞暴露于不同浓度的Art时 ,对肿瘤细胞凋亡的诱导作用 ;用RT PCR、WesternBlotting法 ,检测不同浓度的Art作用于肿瘤细胞时 ,对survivinmRNA和survivin蛋白表达的影响。结果 :HL6 0细胞暴露于Art时 ,呈现典型细胞凋亡特征 ,如 :胞核固缩、形成凋亡小体 ;凋亡细胞的比例呈浓度依赖性增高 ;琼脂糖电泳出现明显的“梯状”条带 ;细胞浆Caspase 3的活性呈浓度依赖性增高等。RT PCR检测表明 ,A5 49细胞暴露于Art 10和 5 0g·L-172h后 ,survivinmRNA的表达呈浓度依赖性降低 ,对照组、10和 5 0mg·L-1处理组的survivin条带和内标GAPDH条带灰度的比值分别为 1.74 5、0 .390和0 .0 2 3;WesternBlotting法也检测到Art抑制Survivin蛋白的表达。结论 :Art诱导肿瘤细胞发生凋亡 ,激活Caspase 3途径 ,可能与抑制survivin基因表达有关     

Effect of actinomycin D on uptake and incorporation of thymidine and hypoxanthine into the acid-soluble and acid-insoluble fractions of rat hepatoma cells grown in culture

Preincubation of MH₁C₁, rat hepatoma cell cultures with actinomycin D between 0.01 μm/ml and 10.0 μm/ml gives a dose-dependent increase in the uptake of thymidine into the acid-soluble fraction up to 400 per cent of controls; the same increase is found in the acid-insoluble fraction. The effect is detectable after 5 min incubation, but is only fully developed after 1–2 hr treatment with the drug. The stimulation could not be blocked by cycloheximide. Preincubation with actinomycin D has little effect on uridine uptake compared to that of thymidine; actinomycin D 1.0 μm/ml after 2 hr increases uridine uptake to 131 per cent of controls. In contrast the uptake of hypoxanthine is inhibited by actinomycin D, 50 per cent inhibition is seen at 0.75 μm/ml. The apparent K_m for the thymidine uptake is 5.9 × 10^?6 M; actinomycin D pretreatment altered the V_max of the reaction but did not change the apparent K_m. The apparent K_m for the hypoxanthine uptake is 5.0 × 10^?6 M; actinomycin D pretreatment gave an apparently noncompetitive inhibition. Actinomycin D does not change the activity of thymidine kinase in homogenates of the cells.     

磺酸基邻苯二甲酰亚氨甲基酞菁锌C光动力效应诱导肿瘤细胞凋亡

目的：研究新型光敏剂磺酸基邻苯二甲酰亚胺甲基酞菁锌Ｃ光动力效应诱导肿瘤细胞凋亡,探索其光动力杀伤机理,方法：用ＡＯ／ＥＢ荧光染色法,流式细胞仪及电子显微镜观察该光敏剂光动力效应与人白血病Ｋ５６２细胞形态,超微结构及ＤＮＡ含量的影响,结果：药物作用２ｈ,以红外照射后继续培养３ｈ以上,就可出现明显的细胞凋亡特性征改变。结论：新型光敏剂磺酸基邻苯二甲酰亚氨甲基酞菁锌Ｃ光动力杀伤作用与诱导肿瘤细胞凋亡有关     

Inhibition of bleomycin-induced [3H] thymidine 5'-triphosphate incorporation into liver and hepatoma nuclei by N-ethyl maleimide and daunomycin.

The addition of bleomycin to a nuclear incorporating system results in an increased incorporation of 3H-thymidine 5'-triphosphate (3H-TTP) into the DNA of liver and hepatoma nuclei. Bleomycin added to the nuclear incorporating system also produces scissions of DNA as determined by sucrose density gradient centrifugation of the extracted DNA. The action of bleomycin is dependent on the presence of sulfhydryl agents in the incubation mixture. Two compounds, N-ethyl maleimide and daunomycin, inhibit the bleomycin-induced incorporation of 3H-TTP preferentially. N-Ethyl maleimide inhibits bleomycin-induced activity in liver and hepatoma 7777 nuclei equally. Lower levels of daunomycin inhibit the bleomycin-induced activity in the hepatoma 7777 nuclei than are required to inhibit the activity in liver nuclei. The two compounds inhibit the bleomycin effect by different mechanisms. The addition of N-ethyl maleimide to bleomycin in the incubation system prevents bleomycin from causing breaks in the DNA. The addition of daunomycin, despite inhibition of bleomycin-induced 3H-TTP incorporation, does not affect the bleomycin-produced breaks in the DNA. N-Ethyl maleimide acts by binding to the DNA and by competing with a sulfhydryl agent for bleomycin-sensitive sites on the DNA. Daunomycin apparently inhibits a repair enzyme that is responsible for the increased incorporation following bleomycin treatment.     

Doxorubicin delivery into tumor cells with ultrasound and microbubbles

Doxorubicin is a potent chemotherapeutic whose severe side effects limit its application. Drug-targeted delivery with noninvasive techniques is required to increase the drug concentration locally and to reduce systemic side effects. Microbubble-assisted ultrasound has become a promising strategy for noninvasive local drug delivery. The aim of this study is to evaluate the applicability and the effectiveness of administration of doxorubicin combined with microbubble-assisted ultrasound in human U-87MG glioblastoma and MDA-MB-231 breast cancer cells. In the present study, the doxorubicin delivery aided by microbubble-assisted ultrasound enhanced the death of breast cancer and glioblastoma cells, including the induction of apoptosis. Various microbubbles were evaluated including Vevo Micromarker, BR14, SonoVue and experimental polymer shelled microbubbles. The results showed that Vevo Micromarker microbubble-assisted ultrasound could induce an enhancement of doxorubicin in glioblastoma and breast cancer cell death. Polylactide-Shelled PEG and Vevo Micromarker microbubbles were the best microbubbles for efficient doxorubicin delivery in the U-87 MG and MDA-MB-231 cells, respectively. Moreover, the induction of apoptosis by doxorubicin and Vevo Micromarker microbubble-assisted ultrasound was examined and results showed a positive increment for acoustic pressures above 600 kPa. The conclusions drawn from in vitro study show the potential of this strategy for an in vivo application.     

Ganoderma lucidum polysaccharides antagonize the suppression on lymphocytes induced by culture supernatants of B16F10 melanoma cells

Objectives Tumour cells produce factors such as interleukin 10 (IL‐10), transforming growth factor β1 (TGF‐β1) and vascular endothelial growth factor (VEGF) that suppress the function of immune cells or induce apoptosis of immune cells. One of the most important goals of tumour immunotherapy is to antagonize this suppression on immune cells. Ganoderma lucidum polysaccharides (Gl‐PS) may have this potential. The purpose of this study was to determine the antagonistic effects of Gl‐PS on the suppression induced by B16F10 melanoma cell culture supernatant (B16F10‐CS) on lymphocytes. Methods Gl‐PS was used on lymphocytes incubated with B16F10‐CS. Enzyme‐linked immunosorbent assay was used to determine the levels of IL‐10, TGF‐β1 and VEGF in B16F10‐CS. The MTT assay was used to determine the proliferation of lymphocytes. Immunocytochemistry and Western blot assay were used to determine perforin and granzyme B production in lymphocytes. Key findings There were elevated levels of IL‐10, TGF‐β1 and VEGF in B16F10‐CS. The lymphocyte proliferation, and perforin and granzyme B production in lymphocytes after induction with phytohemagglutinin, as well as lymphocyte proliferation in the mixed lymphocyte reaction, were suppressed by B16F10‐CS. This suppression was fully or partially antagonized by Gl‐PS. Conclusions B16F10‐CS suppressed lymphocyte proliferation and perforin and granzyme B production in lymphocytes after induction with phytohemagglutinin, as well as lymphocyte proliferation in the mixed lymphocyte reaction. This suppression may be associated with elevated levels of immunosuppressive IL‐10, TGF‐β1 and VEGF in B16F10‐CS. Gl‐PS had antagonistic effects on the immunosuppression induced by B16F10‐CS, suggesting the potential for Gl‐PS in cancer immunotherapy.     

Penetrability of ofloxacin into cultured epithelial cells and macrophages

It is well known that the penetration of drugs into host cells is the minimal requirement to exhibit their efficacy against infections with intracellular bacteria. Thus the penetrability of new quinolones including ofloxacin, norfloxacin and ciprofloxacin was evaluated by comparing their intracellular and extracellular activities by the use of cell infection systems in vitro. It was evidenced that the new quinolones tested were penetrable into both epithelial cells and macrophages, however, ofloxacin was more penetrable than norfloxacin and ciprofloxacin into both types of cells which serve the nest for proliferation of intracellular bacteria.     

Changes in glutathione-redox balance induced by hexachlorocyclohexane and lindane in CHO-K1 cells

1. The basal cytotoxic effect of the organochlorine pesticides hexachlorocyclohexane and lindane on CHO-K1 cultures was assessed at fractions of their lethal doses as determined by the neutral red incorporation (NRI) assay (NRI_6.25, NRI_12.5 and NRI₂₅). The sulphur-redox cycle enzymes glutathione peroxidase, glutathione reductase and glutathione S-transferase, and total and oxidized glutathione were evaluated at several points during the standard growth curve of the cultures. 2. After incubation with each compound for 24h, both glutathione peroxidase and reductase showed a substantial increase at the lowest exposure doses (NRI_6.25) - more significantly for lindane than for 1,2,3,4,5,6-hexachlorocyclohexane (HCH) - and dropped at higher doses of both compounds. The reduced and oxidized glutathione content was greatly diminished at the lower exposures, whereas the total glutathione content was higher at NRI_12.5 values. 3. Changes in cell membrane integrity were assessed for a wide range of pesticide concentrations with the lactate dehydrogenase release assay and lipid peroxidation. Membrane leakage and peroxide production were significantly enhanced at concentrations of HCH 50µg ml^?1, although this effect was not significant at lindane concentrations <200µg ml^?1. 4. Lipid peroxidation increased with exposure to HCH at concentrations as low as NRI_6.25, whereas in the case of lindane, this increase was only significant at doses of NRI₂₅ and above.     

Changes in glutathione-redox balance induced by hexachlorocyclohexane and lindane in CHO-K1 cells

1. The basal cytotoxic effect of the organochlorine pesticides hexachlorocyclohexane and lindane on CHO-K1 cultures was assessed at fractions of their lethal doses as determined by the neutral red incorporation (NRI) assay (NRI(6.25), NRI(12.5) and NRI(25)). The sulphur-redox cycle enzymes glutathione peroxidase, glutathione reductase and glutathione S-transferase, and total and oxidized glutathione were evaluated at several points during the standard growth curve of the cultures. 2. After incubation with each compound for 24 h, both glutathione peroxidase and reductase showed a substantial increase at the lowest exposure doses (NRI(6.25))--more significantly for lindane than for 1,2,3,4,5,6-hexachlorocyclohexane (HCH)--and dropped at higher doses of both compounds. The reduced and oxidized glutathione content was greatly diminished at the lower exposures, whereas the total glutathione content was higher at NRI(12.5) values. 3. Changes in cell membrane integrity were assessed for a wide range of pesticide concentrations with the lactate dehydrogenase release assay and lipid peroxidation. Membrane leakage and peroxide production were significantly enhanced at concentrations of HCH 50 microg ml(-1), although this effect was not significant at lindane concentrations < 200 microg ml(-1). 4. Lipid peroxidation increased with exposure to HCH at concentrations as low as NRI(6.25), whereas in the case of lindane, this increase was only significant at doses of NRI(25) and above.