Design of DNA intercalators to overcome topoisomerase II-mediated multidrug resistance.

Murine P388 (P) leukemia cell lines resistant to amsacrine (P/AMSA), dactinomycin (P/DACT), and doxorubicin (P/DOX) were compared with the parental strain in their sensitivity to a number of derivatives of amsacrine. The P/DACT cell line, which shows the characteristics of a transport-mediated multidrug-resistant cell line, was cross-resistant to vincristine, doxorubicin, etoposide, and a number of acridine-substituted amsacrine derivatives, but was sensitive in vitro and in vivo to amsacrine and its analog CI-921. The P/DOX cell line was cross-resistant to amsacrine but showed a similar pattern of cross-resistance to that of P/DACT in its in vitro response to amsacrine derivatives. In contrast, the P/AMSA line was substantially cross-resistant (from 27- to 146-fold) to all acridine-substituted amsacrine derivatives. However, when the substituents on the anilino side chain of amsacrine were changed, the in vitro cross-resistance of the P/AMSA line could be substantially reduced and even overcome. Derivatives with low cross-resistance ratios were tested in vivo against the P/AMSA leukemia and, in contrast to amsacrine and CI-921, were found to be active. Since the target enzyme for amsacrine action, topoisomerase II, is thought to be structurally modified in the P/AMSA line as well as in some other multidrug-resistant lines, these results suggest the feasibility of tailoring topoisomerase II-directed drugs specifically for the altered enzymes in resistant cells. New drug design approaches are therefore available for overcoming two major types of multidrug resistance.

Effect of flavone acetic acid on Lewis lung carcinoma: evidence for an indirect effect.

Flavone-8-acetic acid (FAA), a new antitumor agent currently undergoing clinical trial, fails to inhibit the growth of early stage Lewis lung (LL) tumors growing in the lung. However, the growth of advanced subcutaneous tumors, arising from inoculation of either the original in vivo LL line or a tissue culture-adapted cell line (LLTC) derived from the LL line was delayed significantly by FAA treatment. Comparison, by clonogenic survival assays, of the cytotoxic effect of FAA on LLTC cells demonstrated that most cell killing occurred between 2 and 8 hours following in vivo exposure but occurred to a much lesser extent and at later times following in vitro exposure. FAA was inactive against LLTC cells growing in vivo in diffusion chambers, suggesting that a host cellular component was necessary for activity. FAA was found to induce hemorrhagic necrosis in the advanced LL tumors, as well as in a number of human tumor xenografts growing in athymic mice. The human cell lines from which the xenografts were derived, as well as the LL tumor lines and P388 leukemia lines, were inhibited by FAA in vitro. However, the ranking of FAA activity in vivo did not parallel that observed in vitro. Together, these observations strongly suggest that FAA has an indirect mode of antitumor action.

Daily supplementation with aged garlic extract, but not raw garlic, protects low density lipoprotein against in vitro oxidation.

The oxidation of low density lipoprotein (LDL) is believed to be an important process in the development and progression of atherosclerosis. In this study, human subjects were supplemented daily with one of: 6 g raw garlic; 2.4 g aged garlic extract (AGE); or 0.8 g DL-alpha-tocopherol acetate for 7 days to determine the effect on the susceptibility of LDL particles to Cu2+-mediated oxidation. LDL isolated from subjects given either alpha-tocopherol or AGE, but not raw garlic, was significantly more resistant to oxidation than LDL isolated from subjects receiving no supplements. These results suggest that if antioxidants are proven to be antiatherogenic, AGE may be useful in preventing atherosclerotic disease.

Responses of bovine T cells to fractionated lysate and culture filtrate proteins of Mycobacterium bovis BCG.

Bacterial cell lysates and culture filtrate proteins of Mycobacterium bovis BCG were each separated in a two-dimensional system that yields soluble protein fractions immediately available for probing with T cells. The fractions were used in lymphocyte proliferation assays using blood lymphocytes from cattle immunized with either viable or gamma-irradiated BCG. Cattle immunized with either form of BCG responded similarly to fractionated lysate proteins. Cattle immunized with viable BCG responded to culture filtrate proteins that were not recognized by cattle immunized with dead BCG. Marked heterogeneity of the responses to the culture filtrate proteins was seen.

A longitudinal serological survey of parvovirus infection in dogs.

Serum samples collected from dogs routinely presented at a clinic between June 1974 and October 1980 were tested for the presence of haemagglutination inhibition (HI) titres to canine parvovirus. The first positive titre (>1:320) was demonstrated in serum collected in October 1979. The first confirmed clinical case of canine parvovirus enteritis was diagnosed by the authors in July 1979. In addition, between 1st December 1980 and 1st March 1981, serum samples were collected from 106 healthy dogs which were presented for canine parvovirus vaccination for the first time. Twenty-four dogs (approx. 23%) showed HI titres >1:320 indicating probable previous canine parvovirus infection. Therefore approx. 80% of dogs in the clinic area were at risk at that time and vaccination should have protected them from infection.

Identification of bovine T cell stimulatory antigens using a cosmid library of Mycobacterium bovis in Mycobacterium smegmatis.

Culture filtrates derived from a Mycobacterium bovis cosmid library in Mycobacterium smegmatis were screened for T cell antigens. Recognition and reactivity were measured by the levels of lymphocyte proliferation and the levels of gamma interferon (IFN-gamma) produced when the culture filtrates were incubated with peripheral blood mononuclear cells (PBMC) taken from cattle immunised with M. bovis BCG. The screening system was optimised to distinguish between M. bovis secreted antigens and normal M. smegmatis secreted proteins. From ten culture filtrates screened, two were identified that induced lymphocyte proliferation and IFN-gamma production. Analysis of the DNA inserts from the recombinant cosmids suggest that they may code for different proteins. The results demonstrate that screening recombinant M. smegmatis culture filtrates can be used to identify M. bovis T cell antigens that are recognised by immunised cattle. These antigens may be important for the development of vaccines with protective ability against bovine tuberculosis.

Comparison of the effects of flavone acetic acid, fostriecin, homoharringtonine and tumour necrosis factor alpha on colon 38 tumours in mice.

Advanced subcutaneous Colon 38 tumours in mice were used for the assessment of activity of a number of anticancer drugs. Activity was measured by histological examination of tumours 24 h after a single dose of the drug and in some cases by tumour growth delay. Agents thought to exert their cytotoxic effect by damaging DNA, including Adriamycin, amsacrine and its analogue CI-921, cyclophosphamide, 5-fluorouracil and methotrexate produced no gross histological changes after 24 h, even though some delayed the growth of subcutaneous tumours. In contrast, flavone acetic acid, fostriecin and homoharringtonine caused extensive necrosis of tumours after 24 h, and each delayed the growth of advanced subcutaneous tumours by at least 10 days when administered as a single dose. The histological effects of flavone acetic acid and fostriecin were indistinguishable from those of recombinant human tumour necrosis factor alpha. It is proposed that histological assay of advanced tumours may provide a useful adjunct to existing methods in screening for antitumour agents with novel mechanisms of action.