Effect of dietary glutamine on tumor glutathione levels and apoptosis-related proteins in DMBA-induced breast cancer of rats

Glutamine (GLN) is a non-essential amino acid that is present in nearly every biochemical pathway and is the major intraorgan nitrogen carrier. GLN via glutamate, is one of the precursors for the synthesis of glutathione (GSH), the major endogenous antioxidant in mammalian cells, which protects them from oxidative injury and cell death. Cancer cells have higher GSH levels than the surrounding normal cells, which attributes to a higher rate of cell proliferation and resistance to chemotherapy. Therefore, selective tumor depletion of GSH presents a promising strategy in cancer treatment. Experimental studies have associated decreased GSH levels with inhibition of proliferation and stimulation of apoptosis. Previous results of our laboratory have provided evidence that dietary GLN diminished tumor development in implantable as well as 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer and elevated GSH in the host tissues. In this study we examined the effects of GLN on GSH levels in DMBA-induced mammary tumors and correlated the results with protein and mRNA expression of apoptosis-related proteins Bcl-2, Bax and caspase-3 in tumor cells. The results have shown that GLN supplementation caused a significant decrease in the tumor GSH levels and the ratio GSH/oxidized GSH (GSSG), accompanied by up-regulation of Bax and caspase-3, and down-regulation of Bcl-2. These findings suggest that dietary GLN supplementation suppresses mammary carcinogenesis by activation of apoptosis in tumor cells and this probably is a result of GSH down-regulation.     

1-Methyl-2-nitrosoimidazole: cytotoxic and glutathione depleting capabilities

We tested 1-methyl-2-nitrosoimidazole (INO), the two electron reduction product of 1-methyl-2-nitroimidazole (INO2) for its in vitro cytotoxicity and glutathione (GSH) depleting capabilities. The half life of INO was shown to be dependent on cell concentration above 10(5) cells/ml, decreasing with increasing cell concentration up to 2 X 10(6) cells/ml. For a 10-fold decrease in cell concentration, from 10(6) to 10(5) cells/ml, the toxicity curve shifted 10-fold towards lower concentrations. At 10(6) cells/ml, INO depleted GSH, in the range of concentrations where toxicity was observed, down to a plateau of 15% of the control level at a concentration of 100 microM INO. Oxidized glutathione (GSSG) levels were not elevated significantly above control cultures at this concentration. INO2, 1000 microM, did not deplete GSH under similar exposure conditions while 2-hydroxylamino-1-methylimidazole (INHOH) depleted GSH minimally at this same concentration. The nitroso intermediate may play a central role in the toxicity and GSH depleting capabilities of 2-nitroimidazoles in mammalian cells.     

人乳腺癌细胞系MCF—7原位移植血管生成超微结构观察及其相关因素表达

目的：探讨人乳腺癌细胞增殖与血管生成方式和时间的超微结构特点,明确人乳腺癌生长中VEGF、F8因子表达水平及其意义,方法：应用人乳腺癌细胞系MCF－7原位移植SCID鼠,光镜、电镜动态观察肿瘤生长与血管生成的细胞形态,结合免疫组化检测VEGF、F8因子的表达水平。结果：人乳腺癌细胞系MCF－7移植后1、2天生存靠固有组织的血管滋养生长,肿瘤组织中未见新生血管生成。肿瘤生长第3天时,肉眼可见周围血管血移植的肿瘤表面延伸,电镜下观察在 存活的癌细胞中可见血管母 细胞出现。第6天时,在癌细胞增生活跃区血管母细胞演化为血管内皮细胞,此时癌细胞群与新生的肿瘤微血管紧密相连。微血管邻旁周边可见极少量的蛋白质网架。第8－10天,内皮细胞有较丰富的线性粒体,粗面内质网增多,池内含有中等量蛋白质,血管内皮的腔面小球增多,光镜下可见肿瘤周围的血管与肿瘤新生血管相通。VEGF、F8因子的表达与肿瘤增殖,侵袭、新生血管生成有关。结论：移植初期人乳腺癌细胞何分泌血管生成因子,肿瘤周围的血管母细胞被激活,快速增殖期癌细胞可诱导血管内上细胞增生形成肿瘤新生血管,逐步演化的肿瘤新生血管与肿瘤边缘固有的血管相连通。     

过表达S100A13基因对甲状腺癌TT细胞增殖特性的影响

背景与目的:有研究表明,S100A13基因与肿瘤的发生有关,而S100A13基因在人甲状腺组织中高表达。本研究旨在探讨S100A13基因过表达对甲状腺癌TT细胞增殖特性的影响。方法:应用Lipofectamine 2000将真核表达载体pCDNA3.1/NT-GFP-S100A13和空载体pCDNA3.1/NT-GFP导入TT细胞,经G418抗性筛选得到稳定的克隆并扩大培养成细胞系,激光共聚焦显微镜观察外源性S100A13蛋白在细胞中的定位。采用real-time RT-PCR和Western blot鉴定稳定过表达S100A13的TT细胞。分别应用细胞生长曲线、流式细胞术方法检测过表达S100A13基因对TT细胞生长速率和细胞周期的影响。结果:成功建立了稳定过表达S100A13和空载体的细胞系TT-S100A13-GFP、TT-GFP。分别将1×104个TT-S100A13-GFP、TT-GFP和TT细胞培养7d后,各组细胞数目分别为(2.30±0.24)×105个、(1.40±0.25)×105个和(1.50±2.20)×105个(P<0.05);TT-S100A13-GFP、TT-GFP和TT细胞经流式细胞仪检测S期的细胞比例分别为(6.47±0.14)%、(5.86±0.23)%和(5.99±0.28)%(P<0.05),G2/M期的细胞比例分别为(50.27±0.66)%、(39.39±0.23)%和(39.64±0.64)%(P<0.05)。结论:过表达S100A13基因对甲状腺癌TT细胞的增殖具有促进作用,促进TT细胞周期从G0/G1期向S期及G2/M期过渡。     

Effect of alpha-interferon, 17 beta-estradiol, and tamoxifen on estrogen receptor concentration and cell cycle kinetics of MCF 7 cells

The interaction of alpha-interferon, 17 beta-estradiol, and tamoxifen on estrogen receptor content, growth fraction, proliferative rate, and total protein synthesis of MCF 7 cells was investigated under culture conditions (minus phenol red and at low concentrations of "stripped" fetal calf serum) allowing for direct stimulation of proliferation by estrogens. Exposure to estradiol alone resulted in a decrease of estrogen receptor content as measured by immunoassay, an increase of the proportion of cells in S phase, and increases in cell proliferation as well as total protein synthesis. alpha-Interferon treatment resulted in cell cycle arrest with reduced proliferation, an increase of estrogen receptor content, but a decrease in the rate of total protein synthesis. Pretreatment with alpha-interferon inhibited the estrogen induced stimulation of cell growth as well as the associated decrease of estrogen receptor content. Tamoxifen treatment resulted in decreased cell proliferation and decrease of estrogen receptor content and of total protein synthesis. These results suggest that the estrogen receptor concentration of MCF 7 cells is growth fraction related. Pretreatment with alpha-interferon enhanced the inhibitory effect of tamoxifen on cell proliferation while preventing the tamoxifen induced reduction of estrogen receptor content. The synergistic effect of alpha-interferon and tamoxifen are most marked following 72 h pretreatment with interferon, when the maximum interferon induced increase of estrogen receptor concentration is evident. The mechanism is thus due probably to an increase of cellular receptor as a ligand for tamoxifen binding and suggests a possible role for the clinical use of interferons combined with tamoxifen.     

Glutathione content in human bone marrow and circadian stage relation to DNA synthesis

DNA synthesis and contents of reduced glutathione (GSH) and oxidized glutathione were determined every 4 hours during a 24-hour period in 70 human bone marrow samples from 10 healthy males. The mean GSH contents during the sampling periods were low, varying from 1.94 to 3.27 nmol/mg protein between the subjects; the mean values for all samples were 2.54 +/- 0.06 nmol/mg protein. The GSH content varied markedly within the individual according to circadian stage (31.0% to 90.2%; mean, 51.4%). Between individuals the mean percentage of cells in DNA synthesis varied from 10.6% to 14.5%, but there was an intraindividual circadian stage-dependent variation, ranging from 48.9% to 274.0% (mean, 126.6%), relative to the lowest value. After adjustment for a slight phase difference between GSH content and DNA synthesis observed for some of the subjects, a statistically significant correlation was found between the GSH content and the fraction of cells in DNA synthesis. The myelosuppressive effect of many chemotherapeutic agents assumed to be detoxified by GSH-dependent mechanism(s) should be considered in the light of the low GSH content in human bone marrow, the circadian variation of DNA synthesis, and the circadian stage-dependent relationship of the GSH content and DNA synthesis.     

Inhibition of cancer cell growth by calcium channel antagonists in the athymic mouse.

The calcium channel antagonists (CCAs) amlodipine, diltiazem, and verapamil inhibited HT-39 human breast cancer cell proliferation in a concentration-dependent manner. The apparent 50% inhibitory dose values were 1.5 microM for the dihydropyridine amlodipine, 5 microM for the benzothiazapine diltiazem, and 10 microM for the phenylalkylamine verapamil. Amlodipine treatment caused a rapid concentration-dependent decrease of intracellular calcium concentration in the HT-39 cell line. Addition of 1 microM amlodipine had no effect on intracellular calcium levels, 3 microM amlodipine lowered intracellular calcium levels in the HT-39 cells by 13.7%, and 10 microM amlodipine lowered intracellular calcium levels by 33.2%. Also, lowering medium calcium levels from 2.0 mM to 0.5 microM resulted in a rapid 41.3% decrease in intracellular calcium and a concomitant 60% inhibition of HT-39 cell DNA synthesis. When HT-39 cells were transplanted into athymic mice, marked hypercalcemia developed. Serum calcium levels from control mice were 8.3 +/- 0.6 mg/dl (mean +/- SE; n = 4); those from tumor-bearing mice were 11.3 +/- 0.08 mg/dl (mean +/- SE; n = 17). Blood calcium levels correlated directly with tumor size (r = 0.91, P less than 0.01). We examined the capacity of three CCAs to specifically inhibit HT-39 tumor growth in vivo. One week after inoculation of HT-39 cells, mice were acclimated to vehicle or 0.1 mg/day amlodipine, 1.0 mg/day diltiazem, or 1.0 mg/day verpamil, in their drinking water, for 7 days. Oral administration of the dihydropyridine amlodipine (0.35 mg/day) for 10 days inhibited HT-39 breast tumor growth by 83.5 +/- 20.1% (mean +/- SE). Oral administration of diltiazem (3.5 mg/day) inhibited HT-39 breast tumor growth rate by 46.5 +/- 6.6% over a 2-week measurement period, and verapamil (3.5 mg/day) inhibited tumor growth rate by 68.2 +/- 9.7% (mean +/- SE). The CCAs had no effect on mouse body weight or gross organ morphology at the concentrations used. Lack of depolarization-induced calcium fluxes in the HT-39 cell line suggests that these cells do not express voltage-operated calcium channels. Thus, our study correlates an effect of amlodipine to lower intracellular calcium levels, by a mechanism not known at present, with its effect to inhibit HT-39 cell proliferation. These findings are important since they demonstrate that amlodipine and other CCAs with known pharmacodynamics and side effects act to blunt breast tumor progression in vivo.     

Determination of intracellular reduced glutathione and glutathione related enzyme activities in cisplatin-sensitive and resistant experimental ovarian carcinoma cell lines

The level of GSH in ovarian carcinoma cells which were sensitive and resistant to cisplatin was serially determined following tumor removal from the animals, in addition, activities of GSH-reductase and GSH-S-transferase were assessed. The GSH level in the resistant line (O-342/DDP) was almost twice as high as that in its sensitive counterpart (O-342), when determined immediately following removal of the tumor (1.55 +/- 0.47 vs. 0.81 +/- 0.32 nmol/10(6) cells). Culturing the cells resulted in a decrease of GSH levels in both cell lines during the first 4 h. Thereafter, GSH levels in both cell lines increased up to 24 h. At this time the GSH level was higher in O-342 than in O-342/DDP. GSH-reductase activity in O-342/DDP cells was significantly higher than in O-342 cells when the enzyme was determined immediately after tumor removal; at the same time there was no difference in activity of GSH-S-transferase between two cell lines. After 24 h in culture, no significant difference between O-342 and O-342/DDP cells could be observed in the activity of the two enzymes.     

Early hematopoietic events during tumor growth in mice

Lewis lung carcinoma (LLC) of C57BL/6 mice, a transplantable tumor widely metastatic by 6-7 days post implant (PI), caused hematopoietic alterations such as progressive anemia (hemoglobin: day 1 PI, 11.0 g/dl; day 19 PI, 7.8 g/dl), neutrophilia (neutrophils: day 1 PI, 2 X 10(3)/microliter; day 19 PI, 22 X 10(3)/microliter), and marrow and splenic myeloid hyperplasia (marrow myeloid-to-erythroid ratio: day 1 PI, 1:1; day 7 PI, 3:1). Accompanying these changes were an increased concentration of marrow granulocyte-macrophage colony-forming units (culture) (GM-CFUC) (day 3 PI, LLC 185 +/- 27% of control; day 19 PI, LLC 265 +/- 10% of control) and accelerated cycling of these myeloid progenitors [day 3 PI, LLC 45.3 +/- 6.5% GM-CFUC in cycle vs. sham (media) injected 17.5 +/- 10.5%; day 7 PI, LLC 52.2 +/- 2.5% vs. sham (media) injected 29.8 +/- 9.8%; day 11 PI, LLC 56.2 +/- 4.4% vs. sham (media) injected 22.2 +/- 14%]. This study questioned whether enhanced hematopoiesis was a result of progressive tumor growth or whether the injection of tumor cells could evoke the response. By use of groups of C57BL/6 mice given an injection of live LLC cells, x-irradiated killed LLC cells, or media, the hematopoietic response to live LLC cells versus dead LLC cells could be dissected. A biphasic colony-stimulating activity (CSA) response in the sera of tumor bearers was found to account for the myelopoietic changes. The first wave of CSA from days 1 to 3 PI stimulated 168 +/- 3.7% more GM-CFUC than control sera and was likely released by dead cells of the tumor inoculum; the second wave from day 7 onward stimulated 220 +/- 7.6% more colonies and was a result of the enlarging tumor mass. Tumor growth was necessary for GM-CFUC proliferation, and the declining growth fraction at day 19 in LLC-bearing mice suggested that hematopoietic exhaustion was a consequence of tumor growth.     

Efficacy of acetylsalicylic acid (aspirin) in skin B16-F0 melanoma tumor-bearing C57BL/6 mice

Several epidemiological studies show that aspirin can act as a chemopreventive agent and decrease the incidences of various cancers including melanoma. In this work, we investigated the in vitro and in vivo efficacy of acetylsalicylic acid (ASA) as an antimelanoma agent in B16-F0 cells and skin B16-F0 melanoma tumor mouse model. Our findings indicate that the IC₅₀ (48 h) for ASA in B16-F0 melanoma cells was 100 μM and that ASA caused a dose- and time-dependent GSH depletion and increase in reactive oxygen species (ROS) formation in B16-F0 melanoma cells. Male C57BL/6 mice were inoculated s.c. with 1?×?10⁶ B16-F0 melanoma cells. ASA (80, 100, and 150 mg/kg) was initiated on day 1 or day 7, or day 9 after cell inoculation and continued daily for 13, 7, and 5 days, respectively. Animals were weighed daily and sacrificed on day 13. The tumors were excised and weighed. The animals receiving 13 days of ASA therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 1?±?12 %, 19?±?22 %, and 50?±?29 %, respectively. Animals receiving 7 days of therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 12?±?14 %, 27?±?14 %, and 40?±?14 %, respectively. No significant tumor growth inhibition was observed with 5 days of therapy. ASA at 100 and 150 mg/kg caused significant tumor growth inhibition in C57BL/6 mice when administered for 13 and 7 days, respectively. The results obtained in this study are consistent with the recent epidemiologically based report that aspirin is associated with lower melanoma risk in humans.     

天然药物莪术醇抑制肿瘤细胞生长及RNA合成影响的初步研究

目的探索莪术之有效活性成分莪术醇对部分肿瘤细胞生长抑制及对RNA合成的影响.方法采用四氮唑盐还原法(MTT)及提取检测RNA等方法,研究天然药物单体莪术醇对13种妇科肿瘤细胞和2种正常乳腺细胞生长抑制及其RNA含量的影响.结果天然药物莪术醇明显抑制MCF7、OV-UL-2、MM231、HeLa细胞的生长(P<0.05);莪术醇抑制肿瘤细胞生长的最佳抑制浓度为50 μg/mL;莪术醇能明显抑制MCF7、MM231、HeLa肿瘤细胞RNA的合成(P<0.05);天然药物莪术醇对正常乳腺细胞MCF12a、MCF10a生长无影响.结论莪术醇在体外能抑制MCF7、MM231、HeLa、OV-UL-2细胞的增殖,并能阻止MCF7、MM231、HeLa细胞RNA的合成.     

Cellular glutathione and thiol measurements from surgically resected human lung tumor and normal lung tissue

Cellular glutathione (GSH) levels were measured from 27 human lung tumor biopsies, enzymatically disaggregated, and compared with cells isolated from normal lung of the same patients. GSH levels from normal lung were similar among patients with a mean value of 11.20 +/- 0.58 (SEM) nmol GSH/mg protein (24 patients) with a range from 6.1 to 17.5 nmol GSH/mg protein. GSH levels varied considerably within and across histological tumor types with the following values: adenocarcinomas, 8.83 +/- 0.96 nmol/mg protein (8 patients); large cell carcinomas, 8.25 +/- 2.51 nmol/mg protein (3 patients); and squamous cell carcinomas, 23.25 +/- 5.99 nmol/mg protein (8 patients). The cyclic GSH reductase assay gave only average GSH values and could not distinguish possible GSH variation among subpopulations of cells isolated. Cell volume measurements and microscopic evaluation of cells isolated from both tumors and normal lung revealed heterogeneity with respect to cell types present. To determine the extent of thiol variation among tumor cell subpopulations, tumor cell suspensions were stained with the thiol-specific stain, monochlorobimane (MCB). The accuracy of MCB staining was tested by flow cytometric analysis of 12 in vitro human tumor cell lines and 3 rodent cell lines. A linear relationship was found between the bimane cellular fluorescence and the cyclic GSH reductase assay for cell lines having less than 80 nmol GSH/mg protein (R2 = 0.82). Above 80 nmol GSH/mg protein the rate of change of the bimane fluorescence intensity with respect to increasing GSH concentrations was much reduced. However, by labeling cells with MCB it was possible to distinguish between cell lines with low versus high GSH content. MCB staining of tumor samples revealed multiple populations of cells with respect to thiol levels. In particular, 2 of 8 squamous cell carcinomas had a proportion of cells with elevated fluorescence intensities (from 10 to 35% of the population) suggesting the presence of cells with greatly elevated thiol levels. These findings underscore the complexity of quantitating intracellular GSH levels from tumor biopsies. The combined use of MCB with flow cytometry and conventional GSH assays may help to delineate subpopulations of cells within tumors with different thiol levels.     

骨巨细胞瘤mdm2、p185、p21及p53免疫组化研究

[目的]探讨mdm2、p185、p21及p53在骨巨细胞瘤（GCT）的表达及与GCT病理分级和复发的关系。[方法]应用SP免疫组织化学方法检测mdm2、p185、p21及p53在52例GCT中的表达（GCT按Jaffe分级：Ⅰ级15例、Ⅱ级25例、Ⅲ级12例）。[结果]52例GCT中mdm2、p185、p21及p53的阳性表达率分别为34.6%（18/52）,21.2%(11/52),13.5%(7/52)及26.f9%(14/52)。不同病理分级阳性表达均无显著性差异。mdm2、p185、p21、p53在复发和无复发的病例中,阳性表达率分别为61.5%(8/13)、25.6%(10/39);38.5(5/13)、15.4%（6/39）;23.1%(3/13)、10.3%(4/29);46.2%(6/13)、20.5%(8/39)。GCT复发与否之间的mdm2表达有显著性差异（P=0.018),而p185、p21及p53的表达无显著性差异。[结论]mdm2、p185、p21及p53在GCT中的表达与病理分级差异无关,而mdm2的表达与其复发与否有关。     

Phorone (diisopropylidene acetone), a glutathione depletor, decreases rat glucocorticoid receptor binding in vivo.

The exact mechanism by which carcinogens and tumor promoters act on the glucocorticoid receptor system in vivo is not known. Based on earlier studies that sulfhydryl-reducing agents stabilize glucocorticoid receptor binding in vitro, some workers have postulated that endogenous reducing factors may be important for glucocorticoid receptor function in vivo. To test whether glutathione (GSH) may serve this purpose, we investigated the effects of phorone, an agent that partially depletes intracellular GSH, on the hepatic cytosolic glucocorticoid receptor (GRc) binding characteristics in intact and 7-10 day adrenalectomized (ADX) adult female Sprague-Dawley rats. Biochemical analysis revealed that a single treatment of phorone (300 mg/kg) to both intact and ADX rats significantly decreased the liver GSH concentration (70-90% of control levels) as well as the GRc maximum binding concentration (30% of control levels). The decrease in GSH levels preceded the reduction in GRc maximum binding concentrations; both effects were reversible after 24 h of treatment. The phorone-mediated decrease of GSH levels was maximum at doses greater than 75 mg/kg, whereas GRc maximum binding concentrations in vivo appeared dose dependent up to 400 mg/kg. Pretreatment with phorone or the carcinogens mirex and 3-methylcholanthrene significantly decreases GRc binding and nuclear uptake in vivo, as well as diminishes intracellular cytosolic GSH levels. Although a temporal relationship between the GSH levels and the GRc maximum binding concentrations in vivo was observed, there was no quantitative relationship between these two parameters based on our phorone dose-response and the carcinogen pretreatment data. Our findings suggest that during the early phases of carcinogenesis, the hepatocellular GSH does not play a direct role upon the biochemical action of certain carcinogens and tumor promoters on the glucocorticoid receptor binding in the liver.     

In-vitro effect of retinoic acid on normal and chronic myeloid leukemia granulopoiesis

The effect of increasing concentrations of retinoic acid (RA) on the in-vitro proliferation of normal and chronic myeloid leukemia (CML) granulo-monocyte precursors (CFU-GM) was studied. 10(-7)M RA added to semisolid cultures stimulated the growth of day 14 but not of day 7 normal CFU-GM, whereas in CML the growth of both populations was either unchanged or inhibited. Five-day and 10-day preincubation of normal bone marrow cells with RA augmented the number of day 14 CFU-GM (by up to 187% with 10(-6) M RA), whereas there was a marked decrease when CML cells were used. Total cellularity was not much affected, though a slight increase in liquid normal bone marrow cultures and a slight fall in CML cultures could be detected. These data point to a difference in the response to RA of normal and CML precursors. They may offer of preclinical basis for its employment to delay the blastic progression of CML.     

Effect of retinoids on the proliferation, morphology and expression of glial fibrillary acidic protein of an anaplastic astrocytoma cell line

We studied the effect of retinoids on the growth and differentiation of a cell line (U 343 MG-A) derived from a human malignant astrocytoma. Cultures treated with all-trans or 13-cis retinoic acid showed a dose-dependent inhibition of proliferation and a marked reduction in the mean cell number at the plateau phase of growth (3.5 x 10(6) vs. 1 x 10(7) cells/25 cm2) compared with untreated cultures. At confluence, cells treated with all-trans or 13-cis retinoic acid were contact-inhibited, whereas control cultures showed crowding, piling, and overgrowth. All-trans retinol or retinyl acetate did not inhibit growth. Astrocytoma cultures treated with all-trans retinoic acid (10(-6) M) for 5 days were modestly growth-inhibited but by day 16 had the same numbers of cells as controls; cultures that received all-trans retinoic acid for 9 days were markedly growth-inhibited for 7 days after the drug was removed. All-trans and 13-cis retinoic acid (10(-6) M) prevented the EDTA-induced cell detachment seen in control cultures. Strongly adherent all-trans retinoic-acid-treated astrocytoma cells grew at a slower rate than did readily detached all-trans retinoic-acid-treated or control cells. Cell spreading, an increased cytoplasmic:nuclear ratio, and greater numbers of broadly bipolar cells, some bearing thin cytoplasmic processes, were seen in cultures treated with 10(-6) M all-trans or 13-cis retinoic acid. Small tightly packed cuboidal cells and large broadly bipolar cells were seen in astrocytoma cultures from which all-trans retinoic acid was removed on days 5 and 9. Indirect immunofluorescence revealed more intense staining with antiserum to glial fibrillary acidic protein in cultures treated with 10(-6) M all-trans retinoic acid than in control cultures; electron-microscope examination of similarly treated cultures revealed more abundant 8-10 nm intermediate filaments than in control cultures. An enzyme-linked immunosorbent assay showed that all-trans or 13-cis retinoic acid caused a dose-dependent increase in the quantity of glial fibrillary acidic protein in the astrocytoma cells.     

Chemosensitization of L-phenylalanine mustard by the thiol-modulating agent buthionine sulfoximine

Glutathione (GSH) plays a crucial role in the protection of normal and tumor tissue against the toxic effects of numerous chemotherapeutic drugs. Therefore, the possible therapeutic benefit of thiol depletion in cancer treatment is dependent upon the relative degree to which tumor or normal tissue is sensitized to the toxic effects of subsequent chemotherapy. To address this issue, the following studies on the chemosensitization of melphalan (L-PAM) by the thiol-depleting agent buthionine sulfoximine (BSO) were conducted in vivo in BDF mice inoculated with L-PAM-resistant murine L1210 leukemia. Different dosing regimens of BSO were found to potentiate L-PAM toxicity in a manner that depended upon the degree of GSH depletion. Multiple i.p. injections of BSO (450 mg/kg every 6 h X 5) were found to reduce GSH concentrations in most tissues by 70-80%, and to decrease the LD50 for L-PAM from 22 to 14 mg/kg. No two organs were found to behave entirely the same with respect to the rate of depletion or recovery of GSH, or to the maximum depletion that could be obtained by BSO. In this regard, the bone marrow was found to be the most resistant tissue to thiol depletion by BSO and was found to tolerate the combination of BSO and therapeutic doses of L-PAM. However, BSO pretreatment markedly inhibited the recovery of the peripheral WBC population at the LD10 dose of L-PAM. Differences also were found in the in vivo metabolism of GSH by L-PAM-sensitive and -resistant murine L1210 leukemia cells. The intracellular concentration of GSH in the resistant cell line was 1.6-fold higher than in the sensitive tumor. Moreover, GSH levels were depleted more rapidly in the resistant tumor relative to the sensitive cell line. A single injection of BSO decreased GSH concentrations in both tumors to equivalent levels (20 nmol/10(7) cells) within 24 h. However, multiple i.p. injections of BSO failed to produce a significant increase in the life-span of L-PAM-treated animals despite a 90% reduction in tumor GSH concentrations (5.5 nmol/10(7) cells). In contrast to the median day survival data, BSO was found to enhance the antitumor activity of L-PAM as determined by an in vivo/in vitro clonogenic assay or by in vivo thymidine incorporation. Using decreased thymidine incorporation as an index of antitumor activity, BSO was found to increase the therapeutic index (LD10/ED50) of L-PAM from 3.6 to 6.5.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of hormones in the growth and regression of human breast cancer cells (MCF-7) transplanted into athymic nude mice

The hormonal environments require by human breast cancer cells MCF-7 to produce solid tumors in nude mice are described. A 100% take was obtained within 7 days following inoculation of 2X10(6) actively growing (log phase) MCF-7 cells into the mammary fat pads of intact, athymic BALB/c nude mice. Tumors failed to develop, even with an inoculum of 20X10(6) cells/mouse, in ovariectomized mice or in mice made diabetic with streptozotocin and observed for 90 days after cell inoculation. A 100% incidence of tumors was obtained in mice that were either hypophysectomized or made diabetic but received injections of 0.2 IU insulin/day/mouse. A 100% incidence of tumors was also obtained in ovariectomized mice that received 17 beta-estradiol in the form of a pellet placed subcutaneously in the interscapular region at the time of cell inoculation. Palpable tumors also developed in ovariectomized mice treated with prolactin, perphenazine, estrone, or estriol, but no takes were observed in ovariectomized mice treated with progesterone, 5 alpha-dihydrotestosterone, or hydrocortisone. Growth of the MCF-7 tumor was stimulated five- to sixfold in both intact and hypophysectomized mice that each received a 17 beta-estradiol pellet. Removal of the 17 beta-estradiol pellets form tumor-bearing ovariectomized mice failed to induce tumor regression. Tumors that continued to grow in ovariectomized mice deprived of 17 beta-estradiol regressed by 50% or more of their initial volume when tamoxifen was injected for 7 days at 5 micrograms/mouse/day) +/- theophyline (1 mg/mouse/day), tumor growth arrest was observed during the 2-to 3-week treatment period. Streptozotocin-induced diabetes in tumor-bearing mice always resulted in complete tumor regression following a 3-week treatment period.     

Tumor necrosis factor-α alters protein metabolism and cell-cycle kinetics in malignant tumor

The effects of tumor necrosis factor-α (TNF) on protein metabolism and cell-cycle kinetics were investigated in malignant tumor. Sprague-Dawley rats, subcutaneously inoculated with Walker 256 carcinosarcoma, were injected intraperitoneally with recombinant human TNF at a dose of 4.75×10⁶ U/kg for 3 consecutive days. Tumor protein metabolism and cell-cycle kinetics were analyzed. The results showed a significant decrease in tumor volume and weight in comparison with control. TNF resulted in significant decrease in tumor protein fractional synthesis rate, protein synthesis and fractional growth rate, but no change of tumor protein fractional degradation rate. TNF also resulted in remarkable decline in labelling index and G1 phase increase of tumor cells, 6 hours after bromodeoxyuridine injection, by cytometry. The results indicated that TNF inhibits tumor growth as a result of decreases in tumor cell DNA and protein syntheses.