A possible role for topoisomerase II in cell death and N-phosphonoacetyl-L-aspartate-resistance frequency and its enhancement by 1-beta-D-arabinofuranosyl cytosine and 5-fluoro-2'-deoxyuridine.

Pretreatment of cells with AraC markedly enhances the frequency of resistance to PALA, methotrexate and 5-fluoro-2'-deoxyuridine (FdUrd) (D.V. De Cicco, A.C. Spradling, Localization of a cis-acting element responsible for the developmentally regulated amplification of Drosophila chorion genes. Cell 38 (1984) 45-54). As a part of studies to elucidate the mechanism for this effect of AraC, the SV40 transformed baby hamster kidney cell line SV28 was treated with either AraC, etoposide or etoposide plus verapamil (to avoid selection for P-glycoprotein-mediated resistance) to isolate cells resistant to AraC or etoposide, respectively. The cells isolated for resistance to AraC (500) were cross-resistant to etoposide and the cells isolated for resistance to etoposide (V5ER and 20ER) were cross-resistant to AraC as well as FdUrd (only V5ER were tested). Enhancement of PALA-resistance frequency by pretreatment with various AraC concentrations and exposure times was greatly attenuated in the three resistant cell lines. Pretreatment with FdUrd markedly enhanced PALA-resistance frequency in SV28 cells, but only weakly did so in V5ER cells. All three resistant cell lines had diminished topoisomerase II as measured by immunoblotting and which was reflected in increased LC50s for etoposide. A comparison of either the etoposide LC50 values or the amount of cellular topoisomerase II, as measured by immunoblotting, with the PALA-resistance frequency in the SV28 and resistant cell lines showed a clear correlation. Increased etoposide LC50 or decreased topoisomerase II correlate with increased PALA-resistance frequency. This holds true for cells treated or not pretreated with AraC. Cells with reduced topoisomerase II are more resistant to the lethal actions of not only etoposide, but also AraC and FdUrd, drugs with different primary sites of action. Cells with reduced topoisomerase II have a higher frequency of resistance to PALA by gene amplification and reduced enhancement of gene amplification frequency when treated with AraC or FdUrd. This suggests two different mechanisms responsible for the increased frequency of resistance and the reduced enhancement of resistance frequency, respectively. These data suggest a role for topoisomerase II in cell death and gene amplification. Possible mechanisms are discussed and a scheme is presented.

Synthesis and evaluation of novel thymidine analogs as antitumor and antiviral agents.

Two series of thymidine analogs with a hydroxyalkylammonium(amine) moiety have been synthesized and evaluated for antitumor and antiviral activities. The hydroxyalkylammonium-(amine) group was introduced at the 5' position of the 2'-deoxyribose residue of thymidine or at a corresponding position in acyclic thymidine analogs. In order to increase the lipophilicity of these compounds and potentially enable them to cross the cell membrane, the free hydroxy group also was esterified with a long hydrocarbon chain. The hexadecanoyl analogs (compounds 1c, 1d, 7c, and 7d) showed moderate antitumor cytotoxicity against SV-28 and KB cell lines (IC50 approximately 20 microM). Compound 1d showed moderate anti-HIV activity (EC50 = 6.8 microM), while compound 5 showed weak anti-HIV activity (EC50 = 55 microM). None of the compounds showed antiherpes simplex virus activity.

The enhancement of the frequency of resistance to N-phosphonoacetyl-L-aspartate and methotrexate by 1-beta-D-arabinofuranosylcytosine: the effect of dipyridamole.

In cultured cells, dipyridamole, in a dose-dependent manner, prevented the enhancement by 1-beta-D-arabinofuranosylcytosine (AraC) of either N-phosphonoacetyl-L-aspartate (PALA)- or methotrexate- resistance frequency. Maximal blockade of enhancement of PALA-resistance frequency occurred if the dipyridamole was added with or within about 20 hr of PALA addition. Thereafter, the effect of dipyridamole decreased. Nitrobenzylthioinosine similarly reduced AraC enhancement of PALA- and methotrexate-resistance frequency. Both dipyridamole and nitrobenzylthioinosine inhibited uridine and thymidine uptake into cells to a similar extent in cells pretreated or not with AraC. Thus, although inhibition of nucleoside uptake would seem a reasonable explanation for the effect on PALA-resistance frequency by dipyridamole, there is no obvious explanation at present of how dipyridamole selectively affects resistance frequency in AraC-pretreated cells.

[Diagnosis and treatment of non-parasitic liver cyst]. / Diagnostika i lechenie neparazitarnoi kisty pecheni.

The experience with treatment of 97 patients with non-parasitary liver cysts by means of percutaneous puncture under guidance of ultrasound investigation and computed tomography has been summarized. The techniques for puncture of non-parasitary liver cysts with drainage and irrigation of a cyst cavity with the solutions of sclerosing substances are presented. The independence of a puncture method of treatment of non-parasitary liver cysts is stressed. A high effectiveness of the method is emphasized.

An analysis of vincristine-resistance in BHK cells pretreated with 1-beta-D-arabinofuranosylcytosine.

As with SV40-transformed BHK cells (1), pretreatment of BHK cells with 1-beta-D-arabinofuranosylcytosine (araC) very markedly enhanced the resistance frequency to N-phosphonoacetyl-L-aspartate (PALA) but only modestly increased the resistance frequency to vincristine (VCR). AraC pretreatment of cells that already had moderate VCR-resistance again only modestly enhanced the resistance frequency at a higher VCR concentration. By contrast, delaying the addition of VCR to BHK cells by 7-24 hours after pretreatment with araC increased the enhancement of VCR-resistance frequency to a level comparable to the enhancement of PALA-resistance frequency. VCR-resistant clones isolated without or after araC pretreatment were analysed for amplification of the multidrug resistance (mdr) gene. One of four clones isolated by single-step selection with no araC pretreatment and four of six clones isolated after araC pretreatment showed amplification of the mdr gene. All VCR-resistant clones tested were cross-resistant to actinomycin D and VCR-resistance was inhibited by treatment with verapamil.

Single-strand breaks in DNA by 1-beta-D-arabinofuranosylcytosine and enhanced resistance frequency to N-phosphonoacetyl-L-aspartate.

1-beta-D-Arabinofuranosylcytosine (araC) caused concentration- and time--dependent single-strand breaks in SV28 cell DNA. Over the same concentration and time range, araC enhances the frequency of resistance to N-phosphonoacetyl-L-aspartate (PALA) and is also cytocidal (1). Comparison of induction of single-strand breaks by araC with its ability either to enhance the frequency of PALA-resistance or to kill cells, however, showed no obvious correlation, whether at different araC concentrations or exposure times. We conclude from these data that if there is a correlation between the ability of araC to cause single-strand breaks and either enhance PALA-resistance frequency or kill cells it is a complicated one or is masked by other events.

1-beta-D-arabinofuranosylcytosine enhancement of resistance to several antineoplastic drugs in mammalian tissue culture cells.

This report describes the enhancement of drug resistance by 1-beta-D-arabinofuranosylcytosine at four individual loci and gene amplification at one locus in a hamster cell line. This drug has been used chemotherapeutically in the treatment of neoplasia and has documented effects on DNA synthesis. Our results in this paper demonstrate that the incidence of resistance to methotrexate, N-(phosphonoacetyl)-L-aspartate, and 5-fluoro-2'-deoxyuridine was appreciably increased after pretreatment with 1-beta-D-arabinofuranosylcytosine and they show that an increase in the incidence of gene amplification is one of the mechanisms by which drug resistance is increased. In contrast, the incidence of vincristine resistance was minimally enhanced by this drug. Possible reasons for this differential enhancement are discussed.

Novel lipid analogs with cytostatic and cytocidal activity.

1-0-Alkyl diol and glyceryl ether lipids with a quaternary ammonium polar head group were synthesized and the cytotoxicity (IC50) tested against KB cells, with low 1-0-alkyl-cleavage activity, and rat hepatoma 77 cells with relatively high 1-0-alkyl-cleavage activity. The original premise was that the compounds would be inactivated by the cleavage enzyme and thus be selectively toxic to cells with less of the enzyme. Results with two other cell lines with equivalent cleavage enzyme, HL-60 and K562-4, however, are not consistent with this premise.

Compounds that inhibit chymotrypsin and cell replication.

Several compounds have been tested for their ability to inhibit bovine pancreatic alpha-chymotrypsin (Ki) and their ability to inhibit cell replication (IC50). There is good agreement over three orders of magnitude between the Ki and the IC50 values of these compounds. The data support the hypothesis that a cellular, chymotrypsin-like activity is necessary for cell replication.

Effect of ethanol on alkaline phosphatase activity in HeLa cells.

Incubation of HeLa cells with 10-80 mM ethanol, caused a dose-dependent decrease in alkaline phosphatase specific activity. With 80 mM ethanol, the decrease became apparent after 24-48 hr incubation. The same dose-dependent effect was observed with the nonmetabolizable alcohol t-butanol. The effect of ethanol was not due to toxicity as the cells continued to replicate for weeks in the presence of ethanol and was reversible. There were no differences in the Km values of alkaline phosphatase in crude extracts or solubilized enzyme prepared either from control or 80 mM ethanol-treated cells. Hydrocortisone and choline chloride, which by themselves increase alkaline phosphatase specific activity, altered the effect of ethanol on alkaline phosphatase. In the presence of either 1 microM hydrocortisone or 40 mM choline chloride, neither 10 nor 20 mM ethanol decreased alkaline phosphatase specific activity.

Inhibition by deoxycytidine, cytidine, and beta-cytosine arabinoside of the induction of alkaline phosphatase activity in HeLa cells.

The main objective of these experiments was the further examination of whether the induction of alkaline phosphatase activity in HeLa cells by 5-iodo-2'-deoxyuridine (IdUrd) depends on the incorporation of IdUrd into DNA. Thymidine (dThd), deoxycytidine (dCyd), cytidine, and beta-cytosine arabinoside (Ara-C) inhibited a dose-dependent manner the induction of alkaline phosphatase activity by IdUrd in HeLa cells, and 5-iodo-2'-deoxycytidine induced activity in a dose-dependent manner at concentrations similar to those of IdUrd. Three of these compounds, dThd, dCyd, and Ara-C, were studied with regard to degree of inhibition of induction and IdUrd incorporation into DNA. Although the various doses of these three compounds decreased the incorporation of IdUrd into DNA, there was no apparent linear correlation between the extent of inhibition of IdUrd incorporation and the degree of inhibition of the induction of alkaline phosphatase activity. dCyd also inhibited a dose-dependent manner the induction of alkaline phosphatase by hydrocortisone, sodium butyrate, and choline chloridee. These results, although not unequivocal, support the idea that IdUrd induction of alkaline phosphatase activity in HeLa cells does not require IdUrd incorporation into DNA. The dCyd altered the thermostability for alkaline phosphatase activity from control or IdUrd-treated cells, and for controls cells the change in thermostability occurred without a change in the enzyme specific activity.

Induction of alkaline phosphatase activity in HeLa cells by sodium butyrate.

The induction of HeLa cell alkaline phosphatase activity by sodium butyrate could be inhibited by the coadministration of caffeine or theophylline. The inhibitions were dose dependent, and at any given concentration the potency was theophylline greater than caffeine. Although the induction by sodium butyrate was more sensitive to the inhibition by the xanthines than was that produced by 5-iodo-2'-deoxyuridine, the magnitudes of the increases in cyclic AMP concentrations after treatment with the xanthines were similar in the inhibition of both types of induction. The induction of alkaline phosphatase activity by sodium butyrate also produced a shift in the thermostability pattern of the enzyme, with a proportionately greater increase in the heat-labile, rather than heat-stable, form of the activity.

The inhibition by xanthine phosphodiesterase inhibitors of the induction of alkaline phosphatase activity in HeLa cells: relationship of enzyme activity to cyclic AMP concentrations.

The three xanthine derivatives, caffeine, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) produced dose-dependent increases in cyclic AMP concentrations in HeLa cells after long term treatment. Only IBMX produced increases over the first 60 minutes, with a peak of approximately 5-fold control values five to 10 minutes after the addition of the drug. About four hours after the addition of either 0.67 or 1.0 mM IBMX there was a second peak in the concentration of cyclic AMP which was at least as large and usually larger than the peak observed at five to ten minutes. Neither caffeine nor theophylline increased cyclic AMP concentrations above control values until one hour after addition of the compounds, and there was no indication of a peak in the concentration at four hours. Between 24 and 72 hours, all three compounds produced elevations in cyclic AMP levels that were steadily maintained. At any given concentration, the order of potency was IBMX greater than theophylline greater than caffeine. If the xanthine derivatives were removed from the medium after 24 hours of treatment, the cyclic AMP concentrations fell to control levels within one hour. Treatment with 5-iodo-2'-deoxyuridine (IdUrd) or hydrocortisone alone did not change the levels of cyclic AMP, nor did the presence of these inducers of alkaline phosphatase activity alter the effects of the xanthine derivations on cyclic AMP concentrations. The data showed a significant correlation between the magnitude of the increase in cycli AMP concentrations over the period from 24 to 72 hours and the degree of inhibition by the xanthine derivatives of the induction of alkaline phosphatase activity.

Induction of alkaline phosphatase activity in HeLa cells by choline chloride.

Choline chloride produced a dose-dependent induction of alkaline phosphatase activity in HeLa cells. At the highest concentration tested, 40 mM, there was a 5- to 7-fold increase in alkaline phosphatase activity, a significantly greater induction than that produced by equiosmolar additions of either NaCl or sucrose. Enzyme activity was higher than control values by 24 hr after the addition of the salt, although the largest increases in activity occurred between 36 and 72 hr. The induction of alkaline phosphatase activity by choline chloride could be inhibited in a dose-dependent manner by the simultaneous addition of either caffeine or theophylline. At comparable concentrations of inhibitor, the magnitude of the inhibition of the induction produced by choline chloride was greater than that observed when the xanthines were used to inhibit the induction by either 5-iodo-2'-deoxyuridine or NaCl. Choline chloride, like NaCl, produced a proportionately greater increase in the heat-stable rather than the heat-labile form of alkaline phosphatase activity.

The incorporation of 5-iodo-2'-deoxyuridine into the DNA of HeLa cells and the induction of alkaline phosphatase activity.

Inhibition of DNA synthesis during the period of exposure of HeLa cells to 5-iodo-2'-deoxyuridine (IUdR) inhibited the induction of alkaline phosphatase activity. This finding, taken together with previous findings that IUdR did not induce alkaline phosphatase activity in the presence of 2-fold molar excess thymidinemonstrated that IUdR incorporation into DNA is correlated with the increase in alkaline phosphatase activity. With the exception of an interim period described in the text, induction of alkaline phosphatase activity was linearly related to medium concentrations of IUdR of up to at least 3 muM. However, the extent of IUdR substitution in DNA did not appear to be related to the degree of enzyme induction. Alkaline phosphatase activity continued to increase at medium concentrations of IUdR from 1 to 3 muM, while little further substitution of DNA occurred.