In vivo modulation of iododeoxyuridine metabolism and incorporation into cellular DNA by 5'-amino-5'-deoxythymidine in normal mouse tissues and two human colon cancer xenografts

This in vivo study examines the ability of 5'-amino-5'-deoxythymidine (5'-AdThd) to modulate 5-iododeoxyuridine (IdUrd) cellular metabolism in two human colon cancer xenografts (HT 29 and HCT-116), two actively proliferating normal mouse tissues (bone marrow and intestine), and a quiescent normal mouse tissue (liver). 5'-AdThd is a thymidine analogue that at low concentrations (<30 micrometer) can increase thymidine kinase activity, which is the rate-limiting enzyme for activation of IdUrd. We reported recently that the in vitro incubation of HT 29 and HCT-116 cells in 5'-AdThd + IdUrd resulted in an enhancement of 5-iodo-2'-dUTP pools, IdUrd DNA incorporation, and subsequent radiosensitization compared with incubation with IdUrd alone (Clin. Cancer Res., 1: 407-416, 1995). These in vitro effects were more significant in the radioresistant cell line HT 29. Using a 6-day continuous infusion of IdUrd (50 or 100 mg/kg/day) and/or 5'-AdThd (200 mg/kg/day), no increase in systemic toxicity (percentage of body weight loss) was observed in athymic nude mice with 5'-AdThd alone or when combined with IdUrd. There was significant dose-dependent, systemic toxicity with IdUrd, which was reversible within 3 days of completing the lower-dose IdUrd infusion. However, a comparison of plasma levels during the 6-day continuous infusion of IdUrd +/- 5'-AdThd showed a significant interaction of IdUrd and 5'-AdThd, resulting in higher plasma levels by day 6 of both compounds and the principal metabolites, iodouracil and deoxyuridine, which is consistent with nonlinear saturating effects on dihydrouracil dehydrogenase. Coadministration of IdUrd and 5'-AdThd resulted in an increase in the percentage of IdUrd DNA incorporation in the two proliferating normal tissues, which was significant only with the lower IdUrd dose. No effect on IdUrd DNA incorporation was found in normal liver at either IdUrd dose +/- 5'-AdThd. Similar to our in vitro data, the continuous infusion of IdUrd and 5'-AdThd showed a significant effect by increasing the percentage of IdUrd DNA incorporation in HT-29 xenografts at both IdUrd doses, whereas coadministration of 5'-AdThd had no such effect in HCT-116 xenografts.     

Role of human liver P450s and cytochrome b5 in the reductive metabolism of 3'-azido-3'-deoxythymidine (AZT) to 3'-amino-3'-deoxythymidine

Our laboratory has shown that human liver microsomes metabolize the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) via a P450-type reductive reaction to a toxic metabolite 3'-amino-3'-deoxythymidine (AMT). In the present study, we examined the role of specific human P450s and other microsomal enzymes in AZT reduction. Under anaerobic conditions in the presence of NADPH, human liver microsomes converted AZT to AMT with kinetics indicative of two enzymatic components, one with a low Km (58-74 microM) and Vmax (107-142 pmol AMT formed/min/mg protein) and the other with a high Km (4.33-5.88 mM) and Vmax (1804-2607 pmol AMT formed/min/mg). Involvement of a specific P450 enzyme in AZT reduction was not detected by using human P450 substrates and inhibitors. Antibodies to human CYP2E1, CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2A6 were also without effect on this reaction. NADH was as effective as NADPH in promoting microsomal AZT reduction, raising the possibility of cytochrome b5 (b5) involvement. Indeed, AZT reduction among six human liver samples correlated strongly with microsomal b5 content (r2 = 0.96) as well as with aggregate P450 content (r2 = 0.97). Upon reconstitution, human liver b5 plus NADH:b5 reductase and CYP2C9 plus NADPH:P450 reductase were both effective catalysts of AZT reduction, which was also supported when CYP2A6 or CYP2E1 was substituted for CYP2C9. Kinetic analysis revealed an AZT Km of 54 microM and Vmax of 301 pmol/min for b5 plus NADH:b5 reductase and an AZT Km of 103 microM and Vmax of 397 pmol/min for CYP2C9 plus NADPH:P450 reductase. Our results indicate that AZT reduction to AMT by human liver microsomes involves both b5 and P450 enzymes plus their corresponding reductases. The capacity of these proteins and b5 to reduce AZT may be a function of their heme prothestic groups.     

Synchronization of cells in the S phase of the cell cycle by 3'-azido-3'-deoxythymidine: implications for cell cytotoxicity

The mechanism of synergy between 3'-azido-3'-deoxythymidine (AZT) and anticancer agents was investigated with emphasis on cell-cycle events. Exposure of exponentially growing WiDr human colon carcinoma cells to AZT resulted in synchronization of cells in the S phase of the cell cycle. Following treatment with AZT at 50 or 200 microM, 62% +/- 3% or 82% +/- 4% of the cells were in the S phase as compared with 36% +/- 2% in the control. Bromodeoxyuridine uptake studies revealed that the synchronized cells actively synthesized DNA. At concentrations of up to 200 microM, AZT produced a cytostatic rather than cytotoxic effect as indicated by viability and cell growth measurements. At 200 microM, AZT-induced synchronization was significant (P = < 0.001) after 12 h of drug exposure, reached a maximum at 24 h, and reversed to baseline levels by 72 h even in the continued presence of the drug. This indicates that AZT-induced cytostasis is a transient and reversible effect. The cell-cycle events seen with AZT in WiDr cells were also observed in eight of nine human tumor cell lines tested. Isobologram analysis of WiDr cells preexposed to AZT for 24 h and then exposed to either AZT-5-fluorouracil or AZT-methotrexate for a further 72 h revealed synergy between AZT and the anticancer agents, indicating that AZT-induced synchronization may have therapeutic benefits.     

Synthesis and antiretroviral evaluation of new alkoxy and aryloxy phosphate derivatives of 3'-azido-3'-deoxythymidine

A series of new ether lipid-3'-azido-3'-deoxythymidine (AZT) conjugates (11a-g) were synthesized and evaluated for anti-HIV activity. The effect of chirality on the antiviral activity was examined through the synthesis of AZT conjugates bearing alkoxypropanols in the lipid portion of the molecule (11a-d). In addition, the long alkyl chain of alkoxyethyl ether lipid-AZT analogs was replaced with aromatic groups (11e-g), and the effect of this structural modification on activity is reported. The results of the biological tests indicate that analogs with a methyl group alpha to the phosphate moiety (11c,d) exhibit a marked degree of stereoselectivity with regard to their anti-HIV activity. Also, replacement of the long alkyl chain with aromatic groups in the oxyalkyl ether phospholipid-AZT conjugates leads to substantially more potent compounds (11e-g) with an anti-HIV activity comparable to that of AZT.     

Intracellular activation and cytotoxicity of three different combinations of 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine

We measured the intracellular activation and cytotoxicity of 3'-azido-3'-deoxythymidine (zidovudine, ZDV) and 2',3'-dideoxyinosine (ddI) when combined at three different clinically relevant combinations of 1:1, 1:10, and 10:1 (ZDV:ddI). The activation of ddI to ddA-TP was increased in all three combinations with ZDV, compared to ddI alone. A maximum twofold increase in ddA-TP was observed, which could not be further increased by raising the concentration of ZDV in the combination. On the other hand, the concentration of ZDV in the combination could be reduced to one-tenth while retaining increased activation of ddI. We also examined the cytotoxicity of these combinations in CEM cells, phytohemagglutinin (PHA)-stimulated and resting human peripheral blood mononuclear cells (PBMCs). CEM cells were the least sensitive overall to the drugs. ZDV showed greater cytotoxicity in stimulated PBMCs than resting PBMCs, whereas the reverse was true for ddI. This could be explained by the different activation pathways of these two drugs. The 1:1 and 10:1 ZDV:ddI combinations showed reduced toxicity compared to the separate drugs. These results indicate that ZDV and ddI need not necessarily be combined together at a ratio of ZDV:ddI of 1:1, but that some alteration in the dosages of ZDV or ddI in patients could be possible without loss of the benefits of combined ZDV:ddI therapy.     

Effect of incorporation of cidofovir into DNA by human cytomegalovirus DNA polymerase on DNA elongation

Cidofovir (CDV) (HPMPC) has potent in vitro and in vivo activity against human cytomegalovirus (HCMV), CDV diphosphate (CDVpp), the putative antiviral metabolite of CDV, is an inhibitor and an alternate substrate of HCMV DNA polymerase. CDV is incorporated with the correct complementation to dGMP in the template, and the incorporated CDV at the primer end is not excised by the 3'-to-5' exonuclease activity of HCMV DNA polymerase. The incorporation of a CDV molecule causes a decrease in the rate of DNA elongation for the addition of the second natural nucleotide from the singly incorporated CDV molecule. The reduction in the rate of DNA (36-mer) synthesis from an 18-mer by one incorporated CDV is 31% that of the control. However, the fidelity of HCMV DNA polymerase is maintained for the addition of the nucleotides following a single incorporated CDV molecule. The rate of DNA synthesis by HCMV DNA polymerase is drastically decreased after the incorporation of two consecutive CDV molecules; the incorporation of a third consecutive CDV molecule is not detectable. Incorporation of two CDV molecules separated by either one or two deoxynucleoside monophosphates (dAMP, dGMP, or dTMP) also drastically decreases the rate of DNA chain elongation by HCMV DNA polymerase. The rate of DNA synthesis decreases by 90% when a template which contains one internally incorporated CDV molecule is used. The inhibition by CDVpp of DNA synthesis by HCMV DNA polymerase and the inability of HCMV DNA polymerase to excise incorporated CDV from DNA may account for the potent and long-lasting anti-CMV activity of CDV.     

Mutagenic consequences of the incorporation of 6-thioguanine into DNA

The stimulatory effect of phytin added to skim milk on acid production of Lactobacillus casei was examined. Phytin stimulated acid production of L. casei fairly well. The stimulatory effect of phytin on acid production was not shown when phytin was treated with Dowex 50 (H+) and neutralized by NaOH solution. The incinerated product of phytin maintained almost equal stimulatory effect on acid production as that before processing. The addition of Mn2+ in the amount contained in a reagent phytin augmented the stimulatory effect on acid production markedly. The further addition of Fe3+, Ca2+, Mg2+ and PO4(3-) in amounts corresponding to their contents in the preparation of phytin as well as Mn2+ increased the effect slightly. The four preparations of phytin contained 0.045-0.20% of Mn, and the greater the Mn content was, the greater the potentiation of acid production.     

Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells

Five cell lines selected for resistance to the cytotoxicity of inhibitors of DNA topoisomerase II have point mutations in the gene that codes for the M(r) 170,000 form of this enzyme. In each case, the mutation results in an amino acid change in or near an ATP binding sequence of the M(r) 170,000 isozyme of topoisomerase II. We used single-strand conformational polymorphism analysis to screen for similar mutations in other drug-resistant cell lines or in leukemic cells from patients previously treated with etoposide or teniposide. We also analyzed the region of the gene that codes for amino acids adjacent to the tyrosine at position 804 of topoisomerase II which binds covalently to DNA. CEM/VM-1, CEM/VM-1-5, and HL-60/AMSA human leukemic cell lines were used as controls; 3 of 3 known mutations were detected by migration differences of polymerase chain reaction products from the RNA extracted from these three lines. A previously unknown mutation was found in the tyrosine 804 region of the M(r) 170,000 topoisomerase II expressed by CEM/VM-1 and CEM/VM-1-5 cells. Sequence analysis showed that substitution of a T for a C at nucleotide 2404 resulted in an amino acid change of a serine for a proline at amino acid 802. No mutations in any of the ATP binding sequences or in the tyrosine 804 region were detected in polymerase chain reaction products from RNA extracted from human leukemia HL-60/MX2 or CEM/MX1 cells (both cell lines selected for resistance to mitoxantrone) or in human myeloma 8226/Dox1V cells (selected for resistance by simultaneous exposure to doxorubicin and verapamil). No mutations were detected in polymerase chain reaction products from RNA extracted from blasts of 15 patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide. We conclude that: (a) single-strand conformational polymorphism analysis is useful for screening for mutations in topoisomerase II; (b) resistance to the cytotoxicity of inhibitors of DNA topoisomerase II is not always associated with mutations in ATP binding sequences or the active site tyrosine region of M(r) 170,000 topoisomerase II; and (c) mutations similar to those detected in drug resistant cells selected in culture have not been identified in blast cells from patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide.     

Effects of chronic ethacrynic acid exposure on glutathione conjugation and MRP expression in human colon tumor cells

This study describes the presence of nitric oxide synthase (NOS-ir) immunoreactive neurons in the thalamic ventrobasal complex of the cat. NOS-ir is co-localized with gamma-aminobutyric acid (GABA-ir) in a subset of small neurons identified as local circuit neurons in previous studies. The double labeled neurons are further identified by a larger soma diameter when compared to GABA-ir only neurons. All NOS-ir somata exhibit GABA-ir but none exhibit immunoreactivity to calbindin.     

Determinants of trimetrexate lethality in human colon cancer cells

We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.     

Quantitative analysis of biological marker synthesis in tumor cell cycle

A quantitative relationship between tumor cell number and biologic marker concentration has been investigated and characterized by the previously developed discrete-time kinetic model for the study of cell kinetics. Here, this model is further expanded to cope with both cell cycle kinetics and biologic marker synthesis. A synchronized tumor-cell population is examined to determine the time course of the synthesis of markers in relation to cell cycle. The DNA content distributions, measured by flow microfluorometry, are analyzed by use of the model, and the cell age distribution is extracted. The average marker content per cell in cell cycle is measured for each time sequence in synchronized cell populations. The model, incorporating the cell age distribution and the average marker content per cell in cell cycle, enables one to generate the marker content distribution, from which the cell number is estimated from the marker concentration. The model performance is further evaluated with Chinese hamster ovary cells, and their polyamine content and the total polyamine concentration during synchronization is calculated and related to the total cell number.     

Atypical microtubule organization in undifferentiated human colon cancer cells

Immunocytochemical identification of GTH I and GTH II cells in the pituitary of the bluefin tuna (Thunnus thynnus) was performed using antisera specific for the common alpha-subunit and the two distinct beta-subunits of tuna (Thunnus obesus) GTH I and GTH II. Cells of the dorsal part of the proximal pars distalis (PPD), in close association with somatotrophs, displayed immunoreactivity of GTHIbeta. GTH IIbeta immunoreactivity was present in cells of the central part of the PPD and the external border of the pars intermedia. Anti-GTHalpha immunostained both GTH Ibeta- and GTH IIbeta-immunoreactive cells and also thyrotrophs. Both GTH Ibeta- and GTH IIbeta-immunoreactive cells were observed in immature bluefin tuna, although there were greater numbers of GTH IIbeta immunoreactive cells. These results suggest that GTH I and GTH II are synthesized in separate cells in the pituitary of the bluefin tuna. The localization and appearance of the two distinct gonadotropic cells of the tuna are compared with the salmonid arrangement.     

Amino acid incorporation into liver and tumor proteins in patients with hepatic neoplasms-effects of liver dearterialization

The effects of liver dearterialization on the rate of amino acid incorporation into liver and tumor proteins were studied with an in vitro method in seven patients with liver metastases. Before liver dearterialization the incorporation rate was 0.074 +/- 0.020 nmol leucine x mg prot-1 x h-1 in liver tissue and 0.234 +/- 0.049 nmol leucine x mg prot-1 x h-1 in tumor tissue. After dearterialization for 1 h the incorporation rate was reduced to about half of the initial values in both liver and tumor tissue. The vascularity of the tumors was evaluated from preoperative hepatic angiograms. The reduction of the incorporation rate was more pronounced in highly vascularized tumors than in poorly vascularized tumors and liver tissue. The clinical implications of a more pronounced metabolic effect of the dearterialization in high vascularized tumors are discussed.