Dose- and schedule-dependent activation and drug synergism between thymidine and 5-aza-2'-deoxycytidine in a human promyelocytic leukemia cell line

The ability of thymidine (dThd) to enhance the metabolism and cytotoxicity of subsequent administered 5-aza-2'-deoxycytidine (5-aza-dCyd) was studied in L1210 cells and in the human promyelocytic leukemic cell line, HL-60. Exposure of L1210 cells to 0.1 mM dThd for 5 h resulted in an increase in the total intracellular and acid-precipitable accumulation of 5-aza-dCyd. Higher dThd concentrations and longer exposure intervals resulted in smaller increments in 5-aza-dCyd accumulation. In contrast, in HL-60 cells, a 24-hr exposure in 1 mM dThd resulted in the greatest intracellular accumulation of 5-aza-dCyd, 3.3 times more accumulation than in control cells. There was also a 4-fold increase in the acid-precipitable accumulation and nearly a 3-fold increase in DNA incorporation of 5-aza-dCyd in HL-60 cells exposed to the same dThd schedule. High-pressure liquid chromatographic analysis demonstrated a greater than 3-fold increase in the intracellular amounts of 5-aza-dCyd metabolites eluting in the triphosphate region in these human cells under identical conditions. Shorter dThd incubation exposure intervals (6 hr) and lower dThd concentration (0.1 mM) produced smaller increments in these studies. Both growth and clonogenic assays of HL-60 cells demonstrated a dose- and schedule sequence-dependent synergism between dThd and 5-aza-dCyd.     

A model to study drug effects on lymphoma and normal cell populations using the AKR/J mouse

The existence of an AKR subline, AKR(Rb6.15)1A1d, with a chromosome marker provided a means to differentiate between proliferating lymphoma and normal cell populations within a single animal. An AKR(Rb6.15)1A1d lymphoma cell line has been maintained for 6 yr by serial passage in AKR/J recipients. The mice die in 7 ± 2.0 days with evidence of extensive infiltration of the tissues by lymphoma cells. Cytogenetic analysis showed that approx. 1% of the metaphase cells in the bone marrow of mice at day 1 of the lymphoma passage were of the AKR(Rb6.15)1A1d donor-type. This increased to 54% by day 4 and 96% by day 6. The number of donor-type metaphase cells per humerus increased from 3.4 ± 0.29 ( × 10³) at day 1 to 2.0 ± 0.49 ( × 10⁵) at day 4 with a concomitant decrease in the number of non-lymphoma host-type metaphase cells. The population doubling time of donor-type metaphase cells per humerus was 1.2 ± 1.4 h. At day 4 there was a significant decrease in the percentage of donor-type metaphase cells in mice that had been treated with BCNU (19.0 ± 5.85%) or spirogermanium (38.6 ± 5.85%) 24 h earlier. For BCNU treated animals, this also represented a decrease to 4.4 ± 1.1( × 10⁴) donor-type metaphase cells per humerus.     

Human amniotic fluid-derived stem cells expressing cytosine deaminase and thymidine kinase inhibits the growth of breast cancer cells in cellular and xenograft mouse models

As human amniotic fluid-derived stem cells (hAFSCs) are capable of multiple lineage differentiation, extensive self-renewal and tumor targeting, they may be valuable for clinical anticancer therapies. In this study, we used hAFSCs as vehicles for targeted delivery of therapeutic suicide genes to breast cancer cells. hAFSCs were engineered to produce AF2.CD-TK cells in order to express two suicide genes encoding bacterial cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) that convert non-toxic prodrugs, 5-fluorocytosine (5-FC) and mono-phosphorylate ganciclovir (GCV-MP), into cytotoxic metabolites, 5-fluorouracil (5-FU) and triphosphate ganciclovir (GCV-TP), respectively. In cell viability test in vitro, AF2.CD-TK cells inhibited the growth of MDA-MB-231 human breast cancer cells in the presence of the 5-FC or GCV prodrugs, or a combination of these two reagents. When the mixture of 5-FC and GCV was treated together, an additive cytotoxic effect was observed in the cell viability. In animal experiments using female BALB/c nude mouse xenografts, which developed by injecting MDA-MB-231 cells, treatment with AF2.CD-TK cells in the presence of 5-FC and GCV significantly reduced tumor volume and weight to the same extent seen in the mice treated with 5-FU. Histopathological and fluorescent staining assays further showed that AF2.CD-TK cells were located exactly at the site of tumor formation. Furthermore, breast tissues treated with AF2.CD-TK cells and two prodrugs maintained their normal structures (for example, the epidermis and reticular layers) while breast tissue structures in 5-FU-treated mice were almost destroyed by the potent cytotoxicity of the drug. Taken together, these results indicate that AF2.CD-TK cells can serve as excellent vehicles in a novel therapeutic cell-based gene-directed prodrug system to selectively target breast malignancies.     

Tumorigenicity of the cyc- variant of the S49 murine lymphoma deficient in the Gs-alpha subunit of adenylate cyclase

S49 cyc- lymphoma cells contain a mutation resulting in loss of a functional guanine nucleotide regulatory protein rendering their adenylate cyclase refractory to most stimuli. S49 wild-type and cyc- clones were used in the present study to investigate the possible association of altered cAMP metabolism with tumorigenicity and metastatic potential. The S49 clones were implanted i.v., i.p., and intracerebrally in both athymic nude mice and syngeneic, immunocompetent BALB/c mice. Both S49 clones gave rise to tumors when inoculated into athymic mice, and no differences were observed in the tumorigenicity or metastatic potential of S49 wild-type and cyc- cells. Implantation of S49 clones in syngeneic BALB/c mice gave rise to few tumors except when administered intracerebrally, where wild-type cells were more tumorigenic than cyc- cells. This raises the possibility of differences in immunogenicity between the S49 clones. Analysis of cell lines derived from tumors grown in athymic mice showed that they retained the phenotype of the S49 clones used for inoculations. The results indicate that, despite differences in adenylate cyclase responsiveness, S49 wild-type and cyc- cells are both highly tumorigenic and metastatic.     

Comparison of different methods to assess the cytotoxic effects of cytosine deaminase and thymidine kinase gene therapy

Dunning R3327 AT-1 rat prostate tumor cells were transfected with a double-fusion suicide gene (CDglyTK) that coded for the cytosine deaminase from E. coli and the thymidine kinase (TK) from HSV-1. The resulting cell line AT-1/CDglyTK was incubated with 10 and 20 microg/ml 5-FC or 0.25 microg/ml GCV, or both 5-FC and GCV 96 hours before harvest. The MTS assay detected cell viabilities of 50+/-5 and 25+/-5% after 5-FC treatment, and 50+/-5% after GCV treatment. The dye exclusion and the colony-forming assay confirmed the data of the MTS assay with GCV (47+/-5 and 32+/-5%), but presented different results for the 5-FC incubation. We detected 100+/-1 and 85+/-5% viable cells after 10 microg/ml 5-FC, and 97+/-1 and 85+/-5% after 20 microg/ml 5-FC treatment, respectively. S-phase arrest in both suicide gene systems was noticeable and a significant increase in cell granularity was observed after incubation with GCV or GCV & 5-FC. This study demonstrates that 5-FC and the metabolized 5-FU act not only as genotoxic reagents, but also as RNA-directed agent, because of the recovery of the cells. On the other hand, a significant S-phase block could be observed after 24 hours incubation with GCV. This short time is enough to incorporate the genotoxic GCV metabolites in the nascent DNA to impair the cell cycle.     

Combination immunotherapy of cancer in a mouse model: synergism between tumor necrosis factor and other defense systems

Bacterial lipopolysaccharide (LPS) induces the release of factors into the serum which enable mice to reject experimental tumors. One such factor is tumor necrosis factor which causes acute necrosis of syngeneic sarcoma transplants in mice. Effective therapeutic use of tumor necrosis factor is limited, however, by its toxicity. We show here that the efficacy of tumor necrosis factor can be substantially increased by combining its application with low doses of LPS. Our data suggest that LPS exerts its antitumor effects by engaging more than one defense mechanism. Characteristic for the activation of a biological system is a concomitant induction of negative feedback mechanisms which antagonize the initial stimulus. Interference with the negative feedback response may substantially increase biological reactions. We show here that the blocking of two negative feedback responses occurring as a consequence of treatment with LPS, namely the production of prostaglandin E and the generation of suppressor T-lymphocytes, increases dramatically the ability of mice to reject tumor transplants. Thus, through appropriate combination of different factors one may reduce the dose of each below toxic levels and through interference with negative feedback responses increase the efficacy of antitumor reagents. We consider our findings in the context of formulating an effective immunotherapy of malignancies and as a promising step toward it.     

Association between MHC class I antigen expression and malignancy of murine T lymphoma variants

Tumor cell variants were derived from an AKR T-cell lymphoma cell line (BW5147, H-2k haplotype). These variants differed in their malignant potential and in their membrane expression of class I MHC antigens. High tumorigenic and spontaneous metastatic capacity was found to be predominantly associated with a decrease of H-2Kk class I major histocompatibility complex (MHC) antigen expression. In contrast, high experimental metastatic capacity correlated strongly with an increased H-2Dk antigen expression. The in vitro invasive potential and the LFA-1 expression of the BW variants showed no correlation with the differential MHC antigen expression and the differential metastatic and tumorigenic capacity of the BW variants. Furthermore, the susceptibility of the BW 5147 variants to TNF and NK-mediated cytotoxicity was not related to the differential metastatic potential and the expression of the class I MHC antigens.     

Potentiation of the direct anticellular activity of mouse interferons: mutual synergism and interferon concentration dependence

Mouse immune (IFN-gamma)2 and virus-type (IFN-alpha/beta) interferons were used separately and in combination in cloning studies with B-16 melanoma cells. IFN-gamma was found to be a more potent mediator of the direct anticellular effect of interferon than was IFN-alpha/beta, as shown not only by a greater sensitivity of B-16 cells to IFN-gamma but also by a steeper slope of the anticellular sensitivity curve of the IFN-gamma. The differences in the slopes of the curves defining their anticellular effect appeared to be inherent in the interferons themselves and not due to an inhibitor of interferon, a stimulator of cell growth, or another factor possessing anticellular activity. The results are consistent with the interpretation that IFN-gamma and IFN-alpha/beta exert their anticellular effects by different mechanisms. The anticellular activity of interferon against B-16 melanoma replication until development was potentiated by mixed preparations of IFN-gamma and IFN-alpha/beta. The potentiation appeared to be an expression of a property of the interferons themselves. Replication units resistant to the potentiated activity of the interferons were not detected. Potentiation levels were dependent on the concentrations of both IFN-gamma and IFN-alpha/beta and continued to increase dramatically as the interferon concentrations increased. Maximum potentiation observed was 214-fold at the highest interferon concentrations used. The data suggested that potentiation is a mutual, synergistic interaction of IFN-gamma and IFN-alpha/beta.     

Physicochemical differences between glucocorticoid-binding components from the corticoid-sensitive and -resistant strains of mouse lymphoma P1798.

The physicochemical properties of nuclear and cytosolic glucocorticoid-binding components from corticoid-sensitive (CS) and corticoid-resistant (CR) mouse lymphoma P1798 cells have been compared. Nuclei or cytosol fractions were prepared from lymphocytes that had been labeled at 37 or 4 degrees, respectively, with 30 nM [3H]triamcinolone acetonide ([3H]TA). [3H]TA was extracted with 0.6 M KCl, 10 mM spermidine, or 4.5 mM MgCl2 from CS nuclei and with 0.6 M KCl or 10 mM spermidine from CR nuclei. As reported previously, nuclear-associated [3H]TA in CR cells was resistant to extraction with mM concentrations of MgCl2. Loss of bound steroid during extraction with 0.6 M KCl was minimized by including the chymotrypsin inhibitor, carbobenzoxy-L-phenylalanine, in the extraction buffer. The inhibitor was not required during extraction with spermidine or MgCl2. Nuclear and cytosolic extracts were examined by analytical agarose gel filtration and glycerol density gradient centrifugation under high salt (0.6 M KCl) conditions. The glucocorticoid-binding component in KCl, spermidine, and MgCl2 extracts from CS nuclei was considerably larger and more asymmetrical [Stokes radius, 57 to 59 A; sedimentation coefficient, 3.64 to 3.70S; molecular weight, 90,000 daltons; frictional ratio, 1.8; axial ratio (prolate ellipsoid), 15] than the [3H]TA-macromolecular complex in KCl and spermidine extracts from CR nuclei[Stokes radius, 29 A; sedimentation coefficient, 3.23 to 3.30S; molecular weight, 40,000 daltons; frictional ratio, 1.25; axial ratio (prolate ellipsoid), 5]. Control experiments showed that the smaller size of the glucocorticoid-binding component in CR nuclei was probably not due to cleavage of a larger, CS-like complex during the extraction procedure. The larger size of the CS [3H]TA complex did not appear to result from aggregation of s a smaller species. No difference in physicochemical parameters of the binding component was observed if cells were labeled with [3H]dexamethasone instead of [3H]TA. However, [3H]dexamethasone complexes were less stable than those formed with [3H]TA as indicated by considerable dissociation of [3H]dexamethasone during gel filtration and gradient centrifugation. This may be due to the 3- to 5-fold lower relative binding affinity of [3H]dexamethasone. Analysis of [3H]TA-labeled cytosol by gel filtration and gradient centrifugation revealed the presence of a single binding component with physicochemical properties similar to those of nuclear [3H]TA complexes from the same strain of tumor. These results suggest that previously described differences in extractability of nuclear-associated [3H]TA between the CS and CR strains of mouse lymphoma P1798 and the lack of response of CR P1798 to glucocorticoid administration may be due, at least in part, to the presence of an altered glucocorticoid-binding component in the resistant tumor cells.     

胃肠道非霍奇金淋巴瘤的治疗

目的：探讨原发于胃肠道的恶性淋巴瘤（Primary Gastrointestinal Malignant Lymphoma PGIML)治疗方式与疗效的关系。方法：回顾性分析我院1981年5月－1997年5月治疗的52例PGIML的资料，将其分为单纯手术（单术）、手术＋化疗（术化）及手术＋放疗＋化疗（综合）三个组，以kaplan-Meier方法分别统计其1、3、5年生存率，Logrank作显著性检验。结果：单术、术化及综合组实际5年生存率分别为20．0％、60．8％及78．5％，与单术相比有显著性意义（P＜0．05），尤以综合组的疗效最好。说明在PGIML治疗中，放疗是一种有效的手段。结论：PGIML治疗宜采用手术配合放、化疗的综合治疗。     

Sequential combination of high-dose cytosine arabinoside and L-asparaginase in the treatment of refractory acute leukemia and malignant lymphoma

Clinical effects of sequential administration of high-dose cytosine arabinoside with L-asparaginase were studied in 5 cases of refractory acute leukemia and 2 cases of non-Hodgkin's lymphoma. A total 12 courses were carried out on these 7 patients and complete remission was obtained in 2 courses and partial remission in 3 courses. Two cases of lymphoma with pleural effusion or CNS invasion achieved partial and complete remission, respectively. The side effects associated with this sequential therapy were nausea, vomiting, diarrhea, fever and conjunctivitis, although these were tolerable. These observations suggest that high-dose cytosine arabinoside combined with L-asparaginase is a useful regimen for refractory leukemia and lymphoma.     

恶性淋巴瘤患者血流感染情况及病原菌耐药性分析

目的 分析恶性淋巴瘤患者血流感染情况及病原菌耐药性.方法 选取310例恶性淋巴瘤患者,检测血流感染的病原菌分布情况,以纸片扩散法进行药敏反应试验.结果 310例恶性淋巴瘤患者中,发生血流感染29例,感染率为9.35％.29例发生血流感染的恶性淋巴瘤患者中共分离出25株病原菌,其中革兰阳性菌15株,占60.00％;革兰阴...     

Application of a new approach for the quantitation of drug synergism to the combination of cis-diamminedichloroplatinum and 1-beta-D-arabinofuranosylcytosine

This report describes the application of a new approach, the universal response surface approach, to the quantitative assessment of drug interaction, i.e., the determination of synergism, antagonism, additivity, potentiation, inhibition, and coalitive action. The specific drug combination and experimental growth system for this introductory application was that of 1-beta-D-arabinofuranosylcytosine (ara-C) and cisplatin with simultaneous drug exposure (1, 3, 6, 12, or 48 h) against L1210 leukemia in vitro. To quantitate the type and degree of drug interaction, a model was fitted using nonlinear regression to the data from each separate experiment, and parameters were estimated (K. C. Syracuse and W. R. Greco, Proc. Biopharm. Sect. Am. Stat. Assoc., 127-132, 1986). The parameters included the maximum cell density over background in absence of drug, the background cell density in presence of infinite drug, the 50% inhibitory concentrations and concentration-effect slopes for each drug, and a synergism-antagonism parameter, alpha. A positive alpha indicates synergism, a negative alpha, antagonism, and a zero alpha, additivity. Maximal synergy was found with a 3-h exposure of ara-C + cisplatin, with alpha = 3.08 +/- 0.96 (SE) and 2.44 +/- 0.70 in two separate experiments. Four different graphic representations of the raw data and fitted curves provide visual indications of goodness of fit of the estimated dose-response surface to the data and visual indications of the intensity of drug interaction. The universal response surface approach is mathematically consistent with the traditional isobologram approach but is more objective, is more quantitative, and is more easily automated. Although specifically developed for in vitro cancer chemotherapy applications, the universal response surface approach should prove to be useful in the fields of pharmacology, toxicology, epidemiology, and biomedical science in general.     

A tumour stage-dependent evolution of drug resistant T cell lymphoma: role of soluble mediators of tumour and host origin

The present investigation was carried out to investigate if soluble mediators present in tumour microenvironment and systemic circulation of a tumour-bearing host can regulate growth properties and response of the cells of a T cell lymphoma to chemotherapeutic drug: cisplatin, depending on the stage of tumour progression. In order to investigate this, tumour cells of a murine T cell lymphoma, designated as Dalton's lymphoma (DL), were incubated in vitro for 48 h in the presence of ascitic fluid and serum obtained from cisplatin treated or untreated tumour hosts at early or late tumour-bearing stages and cell survival was estimated. It was observed that tumour serum and ascitic fluid showed a tumour stage-dependent differential ability to regulate tumour cell survival and susceptibility of the tumour cells to the cytotoxic action of cisplatin. A tumour stage-dependent qualitative and quantitative difference in the profile of cell survival regulating cytokines: IL-1, IL-2, IFN-gamma, TNF-alpha, VEGF and TGF-beta in the ascitic fluid and serum of the tumour-bearing host was observed to be associated with a tumour stage-dependent differential regulation of survival of tumour cells by modulation in the expression of growth regulating proteins: IL-2R, p53, CAD, Hsp70 and Bcl-2. Further the result also showed that production of IL-1, TNF-alpha, and NO by macrophages could be implicated in the differential action of tumour sera on the altered survival responses of tumour cells depending on the stage of tumour growth. Possible mechanisms involved in the tumour stage-dependent differential survival response of tumour cells and evolution of drug resistance are discussed. The finding of this investigation will have clinical implications in designing of therapeutic strategies for T cell lymphoma based on manipulation of tumour growth regulatory mediators present in the tumour microenvironment.     

Significant association between cytotoxic T lymphocyte antigen 4 +49G>A polymorphism and risk of malignant bone tumors

Cytotoxic T lymphocyte antigen 4 (CTLA-4) gene +49G>A polymorphism was implicated to be associated with risk of malignant bone tumors, but the finding was inconclusive owing to the limited sample of a single study. The objective of the current study was to conduct a pooled analysis of four previously published studies to investigate the association between CTLA-4 +49G>A polymorphism and the risk of malignant bone tumors. Data were extracted, and the pooled odds ratio (OR) with the corresponding 95 % confidence interval (95 % CI) was calculated to assess the association. Those four published studies included a total of 2,165 subjects. The pooled results indicated that CTLA-4 +49G>A polymorphism was significantly associated with risk of malignant bone tumors (AA versus GG: OR?=?2.24, 95 % CI 1.67–2.99, P?<?0.001; AA/GA versus GG: OR?=?1.35, 95 % CI 1.14–1.61, P?=?0.001; AA versus GG/GA: OR?=?2.00, 95 % CI 1.53–2.62, P?<?0.001). Stratified analyses by tumor type showed that CTLA-4 +49G>A polymorphism was associated with risks of both osteosarcoma (AA versus GG: OR?=?2.23, 95 % CI 1.45–3.43, P?<?0.001; AA/GA versus GG: OR?=?1.35, 95 % CI 1.04–1.75, P?=?0.024; AA versus GG/GA: OR?=?2.00, 95 % CI 1.34–2.98, P?=?0.001) and Ewing's sarcoma (AA versus GG: OR?=?2.24, 95 % CI 1.51–3.31, P?<?0.001; AA/GA versus GG: OR?=?1.36, 95 % CI 1.07–1.72, P?=?0.011; AA versus GG/GA: OR?=?2.01, 95 % CI 1.39–2.89, P?<?0.001). Therefore, results from the current pooled analysis suggest that CTLA-4 +49G>A polymorphism is associated with risk of malignant bone tumors, including osteosarcoma and Ewing's sarcoma.