Specificity of growth inhibition of melanoma by 4-hydroxyanisole

An experimental study using human melanoma (NEL-MI), rat hepatoma (Fu5-5), and human kidney (293-31) cell lines was undertaken in order to evaluate the antitumor activity of 4-hydroxyanisole (4-OHA) in vitro. Prior reports have indicated highly specific antitumor activity of 4-OHA against melanoma cells in vitro. This specific antitumor activity has been proposed to be due to the oxidation of 4-OHA by tyrosinase to cytotoxic oxidation products. Dose-dependent cytotoxicity was observed when cells were cultured for 72 h in the presence of 4-OHA. At 100 microM, 4-OHA produced growth inhibition of 62%, 32%, and 55% in melanoma, hepatoma, and kidney cell lines, respectively. No effect was seen at 10 microM 4-OHA. 1,000 microM 4-OHA produced 100% kill. Tyrosinase activity was detected only in melanoma cells. The effect of 100 microM 4-OHA on the incorporation of 3H DNA precursors in melanoma, hepatoma, and kidney cells was also studied. Thymidine incorporation was inhibited in all three cell lines at the lowest cell density tested, with the greatest inhibition seen on melanoma cells. As cell density increased, the effect of 4-OHA on thymidine incorporation decreased. With respect to RNA synthesis, 4-OHA significantly reduced the incorporation of uridine in all three cell lines, with the greatest effect in melanoma cells. Cell density also affected the inhibition of uridine incorporation, but to a lesser extent than that observed on thymidine incorporation. The effect of 4-OHA on leucine incorporation was modest and uninfluenced by cell density. Thus, cytotoxicity of 4-OHA may involve two different mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of halofuginone, Wellcome 993 C, oxytetracycline, and diminazene diaceturate on Babesia equi-infected lymphoblastoid cell cultures

The efficacy of halofuginone (DL-trans-7-bromo-6-chloro-3,3-(3-hydroxy-2-piperidyl) acetonyl-4-(3H) quinazolinone), Wellcome 993 C (2-hydroxy-3-cyclohexyl-1,4-naphthoquinone), and oxytetracycline, all of which have been shown to have a schizonticidal effect in the treatment of bovine theileriosis, and the babesicidal drug diminazene diaceturate, were tested against the schizont stages of Babesia equi in cell culture. The in vitro test system measured DNA synthesis in treated and untreated cell lines. Halofuginone (0.02 microgram/ml) and Wellcome 993 C (5 micrograms/ml) suppressed the incorporation of tritiated thymidine by more than 80%. Oxytetracycline was less effective, while diminazene diaceturate showed no notable effect. The insufficient schizonticidal activity may explain the failure of diminazene diaceturate to cure B. equi infections. Schizonticidal drugs either alone, or in combination with known babesicidal drugs, should be tested in the chemotherapy of B. equi infections.     

Feasibility of chemosensitivity testing in soft tissue sarcomas

BACKGROUND: Soft tissue sarcomas comprise less than 1% of all solid malignancies. The presentation and behavior of these tumors differs depending on location and histological characteristics. Standard therapy consists of complete surgical resection in combination with adjuvant radiotherapy. The role of chemotherapy is not clearly defined and is largely restricted to clinical trials. Only a limited number of agents have proved to be effective in soft tissue sarcomas. The use of doxorubicin, epirubicin and ifosfamide allowed response rates of more than 20%. In addition, recent chemotherapy trials did not demonstrate any significant differences in efficacy for various histological subtypes. METHODS: The objective of this study was to gain additional information about the chemosensitivity of soft tissue sarcomas to seven 7 different chemotherapy agents as single drugs and 4 combinations. Therefore we used an established ATP based in-vitro testing system and examined 50 soft tissue sarcomas. Chemosensitivity was assessed using a luciferin-luciferase-based luminescence assay providing individual chemosensitivity indices for each agent tested. RESULTS: The sensitivity varied widely according to the histological subtypes. The tumors state of cellular dedifferentiation played a crucial role for the efficiency of the chemotherapeutic agents. The sensitivity also depended on the presentation of the sarcoma as a primary or recurrent tumor. The highest sensitivity was demonstrated for actinomycin D as a single agent, with 74% of the tumor samples exhibiting a high-grade sensitivity (20% low sensitivity, no resistance). The combination of actinomycin D and ifosfamide yielded a high sensitivity in 76% (2% resistance). Doxorubicin as a mono-therapy or in combination with ifosfamide achieved high sensitivity in 70% and 72%, respectively, and resistance in 6% of the samples. CONCLUSION: Chemosensitivity testing is feasible in soft tissue sarcomas. It can be used to create sensitivity and resistance profiles of established and new cytotoxic agents and their combinations in soft tissue sarcomas. Our data demonstrate measurable discrepancies of the drug efficiency in soft tissue sarcomas, sarcoma subtypes and tumor recurrencies. However, current therapeutic regime does not take this in consideration, yet.     

A comparison of the utilization of thymine and thymidine for the synthesis of DNA-thymine in novikoff hepatoma cells

A comparison was made between the utilization of thymine and thymidine for the synthesis of DNA in Novikoff hepatoma cells growing in suspension culture. When the cell cultures were switched from exponential growth to a relatively non-growing condition, by resuspending them in culture media minus serum for 18 h, there was an 85% decrease in the rate of thymidine incorporation but only a 15% decrease in the rate of thymine incorporation. Exposure to an alkylating agent (methyl methane sulfonate) resulted in a 79% decrease in thymidine incorporation, while thymine incorporation was decreased only 35%. Thymidine at a concentration equal to its Km for incorporation into DNA (4 × 10⁻⁷ M) had virtually no effect on thymine incorporation. It was not until a thymidine concentration of ten times the K_m was employed that appreciable (40%) decreases in the rate of thymine incorporation were observed. Examination of total cellular DNA or nuclear DNA gave similar results. These studies are interpreted as indicating the presence of multiple precursor pools for the synthesis of DNA-thymine in Novikoff hepatoma cells.     

Administration of antibiotics in combination. The antibacterial activity of blood serum and urine after a simultaneous administration of aminoglycosides and ampicillin

Blood serum and urine samples collected from a group of volunteers treated with single doses of ampicillin and aminoglycoside preparations given separately or in combination were tested for their antimicrobial activity against the reference strains Staphylococcus aureus SZK 76/69 and ATCC 6538, Pseudomonas aeruginosa SZK 444 and SZK 385, and Escherichia coli SZK 326/71. Out of all antimicrobials and their combinations tested the most powerful was the combination of netilmicin with ampicillin. Of the therapeutic combinations used nowadays in clinical practice the combined use of gentamicin and ampicillin proved also effective. These antibiotic combinations appear thus to be best suited for the treatment of mixed Pseudomonas aeruginosa and Staphylococcus aureus infections and of urinary tract infections caused by bacterial strains exhibiting in the in vitro susceptibility assays a reduced sensitivity to some of the antibiotic preparations used.     

Selection of chemotherapy for patient treatment utilizing a radiometric versus a cloning system

From the 1950s to the 1970s, a number of in vitro systems that measured inhibition of glucose metabolism were used to predict the responsiveness of patients' tumors to chemotherapy. In vitro-in vivo correlations were excellent, with true positive predictions ranging from 68% to 96% and true negative predictions of 95% to 100%. The radiometric system is a new in vitro technique that measures the conversion of 14C-glucose to 14CO2. The system already has been utilized to screen prospective new antineoplastic agents for cytotoxicity. The present study was undertaken to determine if the radiometric system might be used to predict correctly the responsiveness of an individual patient's tumor to single-agent or combination-agent chemotherapy. Fifty-six tumor specimens were divided and tested for drug sensitivity in the radiometric system and a conventional human tumor clonning system. Overall, there was a significant correlation between in vitro and in vivo results for the conventional cloning system (P = 0.03). However, there was no significant relationship between in vitro and in vivo results for the radiometric system. The radiometric system consistently failed to predict the tumor's clinical sensitivity to single agents. A radiometric system is not useful in predicting the responsiveness of a patient's tumor to single agent chemotherapy and is not a replacement for the more biologically attractive human tumor cloning system.     

The correlation between plasminogen activator activity and thymidine incorporation in mouse bone marrow-derived macrophages : Opposing actions of colony-stimulating factor, phorbol myristate acetate, dexamethasone and prostaglandin E

Plasminogen activator activity and [3H]thymidine incorporation were studied in mouse bone marrow-derived macrophages. The two activities correlated closely in the presence of stimulatory (colony-stimulating factor, phorbol myristate acetate, PMA) and inhibitory (dexamethasone, prostaglandin E1) signals. The actions of dexamethasone and prostaglandin E1 could be overcome by either stimulatory agent, so that the net effect was an alteration in sensitivity of the macrophages to colony-stimulating factor, or PMA. The sensitivity of bone marrow-derived macrophages to CSF-1 was also reduced by the addition of small numbers of CSF-1 unresponsive peritoneal macrophages. Plasminogen activator induction was not a sufficient signal for [3H]thymidine incorporation which requires an additional macromolecular serum component. The serum component was found not to be plasminogen.     

Limitations in the use of [3H]thymidine incorporation into DNA as an indicator of epidermal keratinocyte proliferation in vitro

The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [14C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [3H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

Placental keratinocyte growth factor: partial purification and comparison with epidermal growth factor

A water-soluble extract of term human placenta, which was previously shown to promote proliferative growth of human keratinocytes in defined medium, enhanced both cellular attachment and proliferative growth. We have partially purified the activity which enhanced cell growth and examined its action in keratinocytes. Activity was precipitated from the crude extract by (NH4)2SO4 between 33 and 60% saturation and chromatographed by gel filtration. The activity did not bind to heparin-Sepharose at low ionic strength but was adsorbed to DEAE-cellulose from which it was eluted with NaCl and then passed over phenyl-HPLC to remove bovine serum albumin previously added to protect the activity. The active fraction was applied to gel exclusion HPLC in the presence of 0.02% octyl-beta-D-glucopyranoside, which yielded an apparent Mr 35,000 for the factor. Purification was approximately 200-fold with approximately 4% recovery. The factor appears to be a protein, since activity is destroyed by trypsin. Autoradiography of cultures treated with the placental factor or epidermal growth factor (EGF) revealed that approximately 50% of cells were labeled after treatment with either growth factor compared to 9% in control cultures after a [3H]thymidine pulse. Protein synthesis was increased by about 50% 42 h after treatment with either agent, consistent with a 50% increase in nuclear labeling. Cell number was increased fivefold after 6 days in the presence of the partially purified factor, whereas EGF increased cell number eightfold. Stimulation of [3H]thymidine incorporation by the partially purified factor, in contrast, was about twice that produced by EGF, indicating that thymidine incorporation is preferentially stimulated by the placental factor and does not correlate well with other parameters of proliferative growth. The placental keratinocyte growth factor is a unique factor with a novel effect on incorporation of thymidine into DNA.     

In Vitro Chemotherapeutic Combinations Against Isoniazid-Resistant Mycobacterium tuberculosis and Mycobacterium fortuitum

It is an acceptable medical practice to use second-line antimycobacterial drugs in combination with isoniazid in treatment of isoniazid-resistant tuberculosis. Recent investigations have demonstrated the importance of determining chemotherapeutic interaction in instances of multiple antibiotic use. We studied the inhibitory effect of combinations of isoniazid with ethambutol, rifampin, ethionamide, cycloserine, viomycin, and kanamycin against three isoniazid-resistant strains of Mycobacterium tuberculosis and three strains of M. fortuitum. The isobologram technique with drug concentrations of 0.4 to 100 mug/ml was used. With the exception of single instances in which kanamycin plus isoniazid (M. tuberculosis strain 9999) and ethionamide plus isoniazid (M. fortuitum strain 2080) seemed to have a synergistic effect, neither synergy nor antagonism was noted for any of the combinations. These studies show that the combined use of isoniazid and a second line antimycobacterial agent results in vitro in indifferent inhibitory activity.     

Characterization of the in vitro antimycotic activity of a novel killer protein from Williopsis saturnus DBVPG 4561 against emerging pathogenic yeasts

A novel killer toxin, labelled as KT4561, secreted by Williopsis saturnus DBVPG 4561, was found to possess a wide antimycotic activity against strains of Candida glabrata, Issatchenkia orientalis and Pichia guillermondii. KT4561 was precipitated by ethanol and purified by ion-exchange chromatography. The active protein migrated as a single band in SDS-PAGE and was characterized by a molecular weight of approximately 62 kDa. Purified KT4561 was active across wide ranges of temperature (5-45 degrees C) and pH (4.5-8.0) and displayed a rapid decrease in viability of yeast cells after 4-8 h. The in vitro activity of KT4561 against 102 yeast isolates (79% of clinical origin) was determined: MIC(50) and MIC(90) of strains were 0.08 and 0.15 microg/ml for C. glabrata, 0.03 and 0.23 microg/ml for I. orientalis and 1.50 and 2.25 microg/ml for P. guilliermondii. Comparative susceptibility tests showed that a high number of strains used in the present study were insensitive to selected azole and polyene antibiotics. The present study demonstrated the potential of KT4561 to be applied as novel control agent against pathogenic yeasts.     

88例卵巢上皮性癌行ATP-TCA体外药敏试验结果分析

该研究是探讨三磷酸腺苷生物荧光肿瘤抗癌药物药敏性分析实验（ATP-TCA）在卵巢癌患者化疗中的应用。研究选取88例卵巢上皮性癌新鲜组织行ATP-TCA体外药敏试验,分析结果、计算各种化疗药物敏感性,并与48例对照组患者进行临床近期有效率的比较。结果显示,在体外药敏试验敏感性最强的单药为紫杉醇（51.9%）,敏感性强弱依次为：紫杉醇〉卡铂〉顺铂〉吉西他滨〉拓泊替康〉多西他赛〉依托泊苷〉环磷酰胺〉博来霉素,联合用药方案敏感性较单药增加。药敏组患者临床近期有效率（85.23%）高于对照（68.75%）。ATP-TCA是一种有效的抗癌药物敏感性分析实验,可为卵巢癌患者临床化疗提供个体化的指导方案。     

Mefloquine, an antimalaria drug with antiprion activity in vitro, lacks activity in vivo

In view of the effectiveness of antimalaria drugs inhibiting abnormal protease-resistant prion protein (PrP-res) formation in scrapie agent-infected cells, we tested other antimalarial compounds for similar activity. Mefloquine (MF), a quinoline antimalaria drug, was the most active compound tested against RML and 22L mouse scrapie agent-infected cells, with 50% inhibitory concentrations of approximately 0.5 and approximately 1.2 microM, respectively. However, MF administered to mice did not delay the onset of intraperitoneally inoculated scrapie agent, the result previously observed with quinacrine. While most anti-scrapie agent compounds inhibit PrP-res formation in vitro, many PrP-res inhibitors have no activity in vivo. This underscores the importance of testing promising candidates in vivo.     

Antimutagenicity profiles of some natural substances.

Selected antimutagenicity listings and profiles have been prepared from the literature on the antimutagenicity of retinoids and the carotenoid beta-carotene. The antimutagenicity profiles show: (1) a single antimutagen (e.g., retinol) tested in combination with various mutagens or (2) antimutagens tested against a single mutagen (e.g., aflatoxin B1). Data are presented in the profiles showing a dose range for a given antimutagen and a single dose for the corresponding mutagen; inhibition as well as enhancement of mutagenic activity is indicated. Information was found in the literature on the testing of selected combinations of 16 retinoids and carotenoids vs. 33 mutagens. Of 528 possible antimutagen-mutagen combinations, only 82 (16%) have been evaluated. The most completely evaluated retinoids are retinol (28 mutagens), retinoic acid and retinol acetate (7 mutagens each), and retinal and retinol palmitate (6 mutagens each). beta-Carotene is the most frequently tested carotenoid (15 mutagens). Of the remaining retinoids and carotenoids, 8 were evaluated in combination with a single mutagen and the other 2 were tested against only 2 or 3 mutagens. Most of the data on antimutagenicity in vitro are available for S. typhimurium strains TA98 and TA100. Substantial data also are available for sister-chromatid exchanges in vitro and chromosome aberrations in vitro and in vivo. This report emphasizes the metabolic as well as the antimutagenic effects of retinoids in vitro and in vivo.     

Weak cytotoxic activity of miltefosine against clinical isolates of Acanthamoeba spp

Hexadecylphosphocholine (miltefosine) is an anticancer drug active in vitro against various protozoan parasites, and recently used for the treatment of disseminated Acanthamoeba infection. In the present study, we present results of weak cytotoxic activity of this potential amoebicidal agent for 2 of 3 clinical isolates of Acanthamoeba spp. Although the inhibition effect for all tested concentrations was apparent, and showed 100% eradication of trophozoites of Acanthamoeba castellanii strain at a concentration of 62.5 μM after 24 hr, the strains Acanthamoeba sp. and Acanthamoeba lugdunensis exhibited low sensitivity to hexadecylphosphocholine, even in high concentrations. The determined minimal trophocidal concentrations were 250 μM for Acanthamoeba sp. and 500 μM for A. lugdunensis after 24 hr of exposure. Although hexadecylphosphocholine is a potential agent for treatment of Acanthamoeba keratitis and systemic infections, in clinical practice the possible insusceptibility of the amoebic strain should be considered for optimizing therapy.     

Synergic action between tumor necrosis factor and endotoxins or poly(A·U) on cultured bovine endothelial cells

Summary In order to investigate whether direct effects on tumor vasculature may contribute to induction of necrosis of solid tumors in vivo, agents and combinations with an established different capacity to induce tumor necrosis were studied for their effects on endothelial cells in vitro. Tumor necrosis serum caused a marked inhibition of [³H]thymidine incorporation by bovine umbilical cord endothelial cells after 4h coincubation. Endotoxin was less inhibitory, whereas detoxified endotoxin and recombinant human tumor necrosis factor (rTNF) were hardly active in concentrations that can be achieved in vivo. Combinations of rTNF and (detoxified) endotoxin caused synergic inhibition. By 24h effects of the separate agents and synergic effects of the combinations were much stronger. The nontoxic dsRNA, poly(A·U), also had inhibitory activity, and acted synergistically with rTNF. Morphologically, a combination of endotoxin and rTNF but not the separate constituents induced marked cell detachment by 24 h, an indication of cell death. Whereas both endotoxin and rTNF inhibited DNA synthesis of human endothelial cells, the agents did not act synergistically on these cells. The ability of the agents and the combinations to affect endothelial cells in culture appeared to be well in line with their capacity to induce tumor necrosis. Data suggest that direct (synergic) effects on endothelium may contribute to the induction of vascular damage in tumors by (combinations of) the agents. The fact that endothelial cell death is only induced by the combinations and not by the separate agents in vivo, may be a cause of the greater therapeutic activity of the combinations in vivo. The synergy between rTNF and the other agents indicates that the agents act by different mechanisms.Supported by a grant of the Stichting Koningin Wilhelmina Fonds, Netherlands Cancer Foundation     

Inhibition of herpes simplex thymidine kinase gene expression by DNA methylation is an indirect effect.

The biological activity of in vitro methylated HSV-TK DNA was analysed after microinjection into thymidine kinase negative rat 2 cells. It was found that the fully methylated DNA (Hpa II methylase) was as active as the non methylated control DNA for about 48 hours after injection. DNA reextraction experiments and blot analysis showed that DNA demethylation was not the reason for the observed TK activity. With prolonged cultivation time the methylated DNA becomes rapidly inactive and 100 hrs after injection thymidine incorporation was no longer detectable in the recipient cells. In transformed cells, obtained after coinjection with SV40 DNA, the HSV-DNA was partially demethylated and inactive. Addition of 5-azacytidine to the culture medium induced further demethylation and reactivation of the thymidine kinase gene.     

Interspecific variability in sensitivity to UV radiation and subsequent recovery in selected isolates of marine bacteria

The interspecific variability in the sensitivity of marine bacterial isolates to UV-B (295- to 320-nm) radiation and their ability to recover from previous UV-B stress were examined. Isolates originating from different microenvironments of the northern Adriatic Sea were transferred to aged seawater and exposed to artificial UV-B radiation for 4 h and subsequently to different radiation regimens excluding UV-B to determine the recovery from UV-B stress. Bacterial activity was assessed by thymidine and leucine incorporation measurements prior to and immediately after the exposure to UV-B and after the subsequent exposure to the different radiation regimens. Large interspecific differences among the 11 bacterial isolates were found in the sensitivity to UV-B, ranging from 21 to 92% inhibition of leucine incorporation compared to the bacterial activity measured in dark controls and from 14 to 84% for thymidine incorporation. Interspecific differences in the recovery from the UV stress were also large. An inverse relation was detectable between the ability to recover under dark conditions and the recovery under photosynthetic active radiation (400 to 700 nm). The observed large interspecific differences in the sensitivity to UV-B radiation and even more so in the subsequent recovery from UV-B stress are not related to the prevailing radiation conditions of the microhabitats from which the bacterial isolates originate. Based on our investigations on the 11 marine isolates, we conclude that there are large interspecific differences in the sensitivity to UV-B radiation and even larger differences in the mechanisms of recovery from previous UV stress. This might lead to UV-mediated shifts in the bacterioplankton community composition in marine surface waters.     

Hydroxyurea inhibition of thymidine kinase is dependent on the developmental stage of the brain region

Hydroxyurea, when administered intraperitoneally as three consecutive and varying doses into 7-day-old rats, showed no inhibitory effect on thymidine kinase activity in the cerebral region of the brain. However, when injected intraperitoneally as a single dose into pregnant rats, the activity was found to be significantly inhibited in 16-day-old embryonic cerebrum. The inhibitory effect of the drug was time-dependent. The drug, however, showed no effect on thymidine kinase activity in vitro in either case. On the basis of these results along with our earlier observation regarding the action of hydroxyurea on thymidine kinase activity in 7-day-old cerebellum (1), we conclude that the inhibitory action of hydroxyurea on thymidine kinase activity is dependent on the proliferative stage of cells under study. We further speculate that there may be different forms of thymidine kinase in rapidly dividing and non-dividing cells.