Ascorbic acid and cell survival of adriamycin resistant and sensitive MCF-7 breast tumor cells.

The ability of human cells to regenerate ascorbic acid from dehydroascorbate is partially dependent on the glutathione redox status of the cell and the relative activity of dehydroascorbate reductases. Mammalian dehydroascorbate reductase activity is associated with two proteins known as thioltransferase (glutaredoxin) and protein disulfide isomerase. We compared the specific activity of thioltransferase, protein disulfide isomerase, and other GSH-related enzymes in Adriamycin-resistant human breast tumor cells, MCF-7 ADRR, and Adriamycin-sensitive, MCF-7 WT, tumor cells. MCF-7 ADRR cells had higher activities of glutathione peroxidase (34.7 fold), nonseleno-glutathione peroxidase (glutathione S-transferases; 5.3 fold), thioredoxin (2.3 fold), and thioltransferase (4.0 fold) compared with the WT Adriamycin-sensitive cell line. Thioltransferase was detected in Western blots in extracts of ADRR MCF-7 cells but not in WT MCF-7 cells. alpha-Tocopherol in the membrane and cytosolic fractions was 2.8 and 3.0 fold higher, respectively, in Adriamycin-resistant compared with Adriamycin-sensitive cells. Supplementation of MCF-7 cells with L-ascorbic acid 2-phosphate (2 and 10 mM) had no effect on WT cell viability after 5 days incubation with up to 0.33 microM Adriamycin. In contrast, supplementation of ADRR MCF-7 cells with L-ascorbic acid 2-phosphate resulted in enhanced resistance up to 3.4 microM Adriamycin over a 5-day incubation. Both lines of MCF-7 cells demonstrated the ability to utilize ascorbic acid as the 2-phosphate derivative. After 48 h incubation with 8.6 microM Adriamycin, the resistant cells maintained normal viability and ascorbate-dehydroascorbate levels, whereas drug-sensitive cells had significantly lower ascorbate with a higher percent dehydroascorbate and increased cell death as judged by cell protein levels (52% of controls).

Evaluation of nuclear morphology in adriamycin sensitive and resistant cells.

The adriamycin sensitive and resistant human breast cancer cells (MCF-7) were investigated by computerized image analysis for establishment of characteristic cell nuclei-morphological differences. Using specially developed algorithms and a large set of subvisual parameters, morphological features characteristic for the resistant cell line were described. The most useful parameters for distinguishing the resistant from sensitive cells are related to chromatin structure and include integral optical density per micron 2, the average area of chromatin region, and the integral density of the chromatin region per micron 2. Besides the size of the nuclei, the two features with the most discriminatory power belonged to those characterizing patterns of chromatin condensation. The results of quantitative analysis suggest that drug resistance relates to specific chromatin patterns. Compared to sensitive cell line, the resistant MCF-7 cells show distinguished coarse chromatin pattern with enlarged condensed chromatin regions.

A phase II trial of intermittent leukocyte interferon and high dose chlorambucil in the treatment of non-Hodgkin's lymphoma resistant to conventional therapy.

Thirteen patients with advanced non-Hodgkin's lymphoma who previously failed conventional chemotherapy protocols were treated with a combination of alpha-interferon (IFN) 6,000,000 units i.m. days 1-5 and 8, plus chlorambucil (CLB) 16 mg/m2 days 5-9 repeated every 4 weeks. There were five complete responses (CRs) and one partial response (PR) (46% total responses) with mean duration of remission of 456+ days. Responses were obtained in low and intermediate grade lymphomas. Toxicity was acceptable and easily managed. It is unlikely that IFN alone using this low dose intermittent schedule is responsible for the remissions. The combination of the two agents appears to be an effective treatment modality. IFN may be functioning as a biological response modifier when used in combination with a cytotoxic agent.

The effect of selenium on phagocytosis in humans.

The effect of selenium on immune responses in animals and humans is controversial. It has been reported that phagocytosis as a part of the immune function is affected by selenium deficiency. We conducted a study to investigate the effect of selenium on the phagocytic function of polymorphonuclear leukocytes (PMNs) in normal healthy individuals before and after selenium supplementation. Ingestion of sodium selenite 400 micrograms/day (182.8 micrograms pure selenium) resulted in a significant increase in plasma selenium levels. The phagocytic function of PMNs was measured by ingestion of Oil Red O paraffin droplets and chemiluminescence tests. The phagocytic function was increased, but the results before and after selenium supplementation were not significant. It was concluded that inorganic selenium was not an efficient stimulating agent of phagocytosis in humans.

Comparative prevalence of rabies antibodies among household and unclaimed/stray dogs as determined by the immune adherence haemagglutination assay.

The immune adherence haemagglutination assay (IAHA), widely used for human viral disease diagnosis, has been adapted for detection of rabies virus antibodies in dog sera. Rabies virus antibody titres obtained by the IAHA correlated well with those obtained by the currently accepted test for rabies antibody determination, the rapid-fluorescent-focus-inhibition test (RFFIT). Although it is not known if the antibodies detected in IAHA test represent neutralizing antibodies against rabies, IAHA has several advantages over the RFFIT: the IAHA is rapid, requiring about seven hours for results to be available; it is relatively inexpensive and easy to perform; uses reagents commonly available in any routine virology laboratory; and uses inactivated rabies virus, thus eliminating hazards associated with the use of live virus in RFFIT. Using this test we found that rabies antibody titres were significantly higher, and at the same time more prevalent, among household dogs than among the unclaimed/stray dogs. The results re-emphasize the increased hazard associated with unclaimed/stray dogs and the need for vaccination of all dogs.

Abrogation of adriamycin-induced cardiotoxicity by selenium in rabbits.

Adriamycin-induced cardiomyopathy in rabbits was produced by intravenous injections of the drug with a short therapeutic schedule (3 mg/kg body wt administered as four intermittent doses). Animals receiving selenium supplementation of Adriamycin showed preservation of the normal pattern of the heart histologic picture. The protective effect of selenium was accompanied by increased selenium levels in the plasma and the heart muscle. An eventual interaction between the antitumor effect of Adriamycin and the protective effect of selenium was ruled out by in vitro experiments using the L1210 cell line. Selenium did not abrogate the antiproliferative effect of Adriamycin when the cells were treated simultaneously with both agents. The results from this study indicate that Adriamycin-induced cardiotoxicity could be prevented by selenium if the animals were pretreated with selenium, rather than simultaneous administration of both agents. The mechanism of this effect is not entirely understood.

Changes in calcium channel activity in membranes from cis-diammine-dichloroplatinum(II)-resistant and -sensitive L1210 cells.

Ca2+ channels from lipid and proteolipid fractions of cisplatin-sensitive and cisplatin-resistant cells were reconstituted and characterized in bilayer lipid membranes formed at the tips of patch-clamp micropipets. The characteristics of the Ca2+ channels were typical for the endoplasmic reticulum membrane channel activity. They had a relatively large unit conductance and were modified by typical activators (nucleotides) and inhibitors (ruthenium red, verapamil). Different doses of nifedipine did not inhibit Ca2+ channel activity. A substantial difference between the single-channel properties of the two types of investigated membranes was observed. The mean open time and the open state probability of channels reconstituted in bilayer lipid membranes from the membrane components of cisplatin-resistant cells were larger than those in bilayer lipid membranes made from components of cisplatin-sensitive cells. Ruthenium red (7 X 10(-7) M) inhibited the channel activity in both types of membranes to the same level. The observed effects could be related to an increased Ca2+ release from the intracellular Ca2+ stores (endoplasmic reticulum system) accompanied by an enhanced intracytoplasmic Ca2+ concentration in cisplatin-resistant cells. These changes in the Ca2+ concentration level may be responsible for the higher antitumor drug efflux rate and the development of the drug resistance. The suggestion is made that specific inhibitors of the Ca2+ transport across the membranes of the subcellular Ca2+-storing organelles may be tested as agents for overcoming the antitumor drug resistance.

Chemotherapeutic agents and modulation of natural killer cell activity in vitro.

This study investigated the effect of various clinically used chemotherapeutic agents on non-modulated and human alpha Interferon (IFN) modulated natural killer cell (NK) activity. The inhibitors of DNA synthesis, Adriamycin, Cis-Platinum, 5-Fluorouracil and Methotrexate, did not alter NK cell activity at comparable clinically used doses. Similarly, Vincristine and Vinblastine, which are antimitotic agents, did not affect the NK activity. Only inhibitors of RNA synthesis L-Asparaginase and Actinomycin D reduced non-modulated and IFN modulated NK cell activity.

Leukocyte interferon as a possible biological response modifier in lymphoproliferative disorders resistant to standard therapy.

In vitro and in vivo studies utilizing a combination of leukocyte interferon-alpha (IFN) and chlorambucil (CLB) were done to investigate possible synergism between a biological response modifier and a chemotherapy drug. In vitro studies utilized a human myeloid leukemia cell line (K-562) pretreated with IFN and then exposed to CLB. The combination resulted in significant depression of cell growth compared with use of IFN or CLB alone. In vivo studies involved eight heavily pretreated patients given 6 million units IFN for 5 days followed by oral CLB (16 mg/m2) for 5 days repeated every 4 weeks. Three myeloma patients had reduction in immunoglobulins and experienced clinical responses. Three of four patients with Hodgkin's disease responded after relatively short periods of treatment. One patient with a diffuse lymphocytic lymphoma had a complete unmaintained remission lasting 6 months. Toxicity was minimal, with mild fever, nausea, and vomiting. These preliminary studies suggest that IFN may be a biological response modifier when used in combination with a cytotoxic agent.

Rapid test for detection of rabies antibodies in human serum.

A simple, sensitive, rapid method based on the principle of immunoadherence hemagglutination (IAHA) has been devised for the detection of rabies antibody. In this test, fixation of complement to complexes of rabies antigen with specific antibodies is readily detected by agglutination of human erythrocytes bearing receptors for C3. Sera from individuals undergoing preexposure rabies immunization were tested for rabies antibodies by the IAHA method and by a virus neutralization test performed in tissue culture, the rapid fluorescent focus inhibition test. IAHA titers showed a high degree of correlation with rapid fluorescent focus inhibition test titers, although it is not known whether results of the IAHA test represent the detection of neutralizing antibodies. An advantage of the IAHA test over the rapid fluorescent focus inhibition test was that results were obtained in a shorter period of time. In some instances, this can be of clinical significance in determining antibody levels to rabies virus. Furthermore, the IAHA test is most applicable as a rapid screening tool for the detection and quantitation of rabies antibodies in vaccinated subjects.

Biological characterization of an Epstein-Barr nuclear antigen-positive American Burkitt's tumor-derived cell line.

Two distinct cultures derived from a lymphoid cell line designated NAB were characterized immunologically, morphologically, and cytogenetically. Both cultures were positive for Epstein-Barr nuclear antigen. NAB I cultures were negative for virus capsid antigen and early antigen and were not affected by treatment with 5-iododeoxyurdine. NAB II cultures were positive for virus capsid antigen and early antigen, which increased with 5-iododeoxyuridine treatment. Both cultures were superinfected with virus prepared from P3HR-1 cells. Cell-free virus concentrates prepared from both cultures were inactive for transformation and infectivity. NAB I and NAB II cells were lymphoid as determined by light and electron microscopy. NAB II cells showed morphological alterations characteristic of herpes infection. 5-iododeoxyuridine-treated cells from both cultures revealed ultrastructural characteristics of cells infected with herpes-viruses but without particles. In addition, the induction of tubuloreticular structures within the endoplasmic reticulum was observed. Cytogenetic analysis of both cultures revealed a rearranged chromosome 14 and several other chromosome aberrations, three of which may be used as a reliable means of identifying NAB cultures.

Production of infectious Epstein-Barr virus by using an intermittent culture collection system.

Procedures were developed to produce infectious Epstein-Barr virus in large glass fermentors with limited mechanical agitation by using an intermittent culture collection system. The system appears quite suitable for use in fermentation vessels of 50 liters or larger.