Azido-3'-deoxythymidine Incorporation Research Articles

Genetic alterations in K‐ras and p53 cancer genes in lung neoplasms from Swiss (CD‐1) male mice exposed transplacentally to AZT

A transplacental carcinogenicity study was conducted by exposing pregnant Swiss (CD-1) mice to 0, 50, 100, 200, or 300 mg of 3'-azido-3'-deoxythymidine (AZT)/kg bw/day, through a 18 to 19-day gestation [National Toxicology Program, NIH Pub. No. 04-4458, 2004]. The incidences of alveolar/bronchiolar adenomas and carcinomas, in the 200 and 300 mg/kg male treatment groups, were significantly greater than that of the controls. In the present study, we evaluated the benign and malignant lung neoplasms from this bioassay for point mutations, in the K-ras and p53 cancer genes that are often mutated in human lung tumors. K-ras and p53 mutations were detected by cycle sequencing of polymerase chain reaction-amplified DNA, isolated from formalin-fixed, paraffin-embedded neoplasms. K-ras mutations were detected in 25 of 38 (66%) of the AZT-induced lung tumors, and the predominant mutations were codon 12 G-->T transversions. p53 mutations were detected in 32 of 38 (84%) of the AZT-induced lung tumors, with the predominant mutations being exon 8, codon 285 A-->T transversions, and exon 6, codon 198 T-->A transversions. No K-ras or p53 mutations were detected in five tumors, examined from control mice. The patterns of mutations identified in the lung tumors suggest that incorporation of AZT or its metabolites into DNA, oxidative stress, and genomic instability may be the contributing factors to the mutation profile and development of lung cancer in these mice.

Plasma drug levels compared with DNA incorporation of 3'-azido-3'-deoxythymidine (AZT) in adult cynomolgus (Macaca fascicularis) monkeys.

Zidovudine (3'-azido-3'-deoxythymidine, AZT), widely used for the therapy of the Human Immunodeficiency Virus-1 (HIV-1), is a nucleoside analog of thymidine that becomes phosphorylated and incorporated into nuclear and mitochondrial DNA. Levels of AZT incorporation into DNA of humans, monkeys, and mice are highly variable and suggest interindividual variability in phosphorylation pathways. In addition, studies in rhesus monkeys (1) have shown a lack of correlation between levels of unbound AZT in plasma and tissue AZT-DNA. However, the correlation between plasma AZT and tissue AZT-DNA has not been previously examined in the same primate. Here we examine the relationship between AZT-DNA incorporation in leukocytes and multiple organs, and levels of the drug circulating in plasma of adult female cynomolgus (Macaca fascicularis) monkeys. Three monkeys were dosed with 40.0 mg of AZT/day for 30 days by naso-gastric intubation. The average daily dose of 9.9 mg of AZT/kg/body wt was similar to the approximately 8.6 mg of AZT/kg/body wt (600 mg/day) given to adult HIV-1-infected patients. In all three monkeys, at the time of sampling, values for AZT concentrations in plasma were similar and values for AZT incorporation into leukocyte DNA (86.1, 100.0, and 114.1 molecules of AZT/10(6) nucleotides) were also similar. AZT-DNA incorporation was detected in liver, uterus, spleen, and kidney from the three AZT-exposed animals, with values for positive samples ranging from 5.8 to 97.4 molecules of AZT/10(6) nucleotides. In brain cortex and lung DNA from AZT-exposed animals, AZT incorporation was undetectable. The data suggest that organ-specific differences in AZT uptake and/or metabolism may contribute to AZT phosphorylation and subsequent drug incorporation into DNA. In addition, AZT-DNA levels in monkey organs were similar to or lower than values observed in peripheral leukocytes of adult AIDS patients.

Enhanced delivery of AZT to macrophages via acetylated LDL

It is known that infected macrophages play an important role in HIV pathogenesis acting as a reservoir for dissemination of the virus to various organs. Enhanced and/or specific delivery of anti-HIV agents to infected cells is expected to improve their therapeutic index by increasing efficacy and reducing toxicity. Acetylated low density lipoproteins (AcLDL) are known to be taken up by macrophages via scavenger receptors and appear to be good carriers for targeting drug molecules to macrophages. This study investigated the delivery of 3′-azido-3′-deoxythymidine (AZT), an anti-HIV agent, to macrophages using AcLDL. Since the incorporation of AZT into AcLDL was found to be low, several derivatives of AZT including 5′- O-13-oxamyristate-AZT (5′- O-oxaMyr-AZT) have been synthesized as prodrugs. The prodrugs were incorporated into AcLDL using two different methods, namely the contact method and the microemulsion method. Our results demonstrated that the microemulsion method was more effective. The physicochemical properties of the AcLDL/prodrug complex were evaluated by electrophoresis and electron microscopy (EM). Incubation of the complex with plasma resulted in little distribution of the incorporated drug molecules from AcLDL to other components of the plasma, suggesting that the complex was quite stable. Cellular uptake studies using J774.A and U937 demonstrated that AcLDL/prodrug was taken up about 10 times more than AZT. The presence of excess AcLDL was found to inhibit the cellular uptake of AcLDL/5′- O-oxaMyr-AZT by macrophages while excess high density lipoprotein (HDL) or low density lipoprotein (LDL) was found to have little effect, suggesting that the AcLDL/prodrug complex is taken up into macrophages via the scavenger receptor.

Incorporation of zidovudine into cord blood DNA of infants and peripheral blood DNA of their HIV-1-positive mothers.

The nucleoside analogue 3'-azido-3'-deoxythymidine (AZT) is a weak carcinogen in adult female mice and a moderately strong carcinogen in the offspring of female mice given the drug during gestation. In addition, incorporation of AZT into DNA was observed in multiple organs of transplacentally exposed newborn mice. Here we investigate the incorporation of AZT into peripheral leukocyte DNA of HIV-1-positive adult pregnant women given AZT for variable times during gestation and cord blood of infants exposed to AZT in utero. The length of treatment varied between 10 days and 9 months. High molecular weight DNA was extracted from maternal peripheral blood mononuclear cells (PBMC) and infant cord blood. A specific AZT-DNA radioimmunoassay was used to determine the amount of AZT incorporated into leukocyte DNA. Incorporation of AZT into DNA ranged up to 183.3 and 344.5 molecules of AZT/10(6) nucleotides in the mothers and infants, respectively, and was detected in about 70% of samples. Therefore, AZT-induced mutagenic events are possible in the majority of adults and infants. No correlation was found between level of incorporation and length of AZT treatment, suggesting that the differences observed among the individuals arise from variability in AZT metabolism. These data support previous observations that a high degree of inter-individual variability in AZT phosphorylation occurs in primates.

Mutagenicity and loss of heterozygosity at the APRT locus in human lymphoblastoid cells exposed to 3'-azido-3'-deoxythymidine.

Previous experiments in our research group showed that 3'-azido-3'-deoxythymidine (AZT) caused increased mutant frequencies (Mfs) at the X-linked hypoxanthine-guanine phosphoribosyltransferase (HPRT) and the autosomal thymidine kinase (TK) genes in human lymphoblastoid cells and that there was a significant positive correlation between AZT incorporation into cellular DNA and AZT-induced TK Mfs. In the current study, the mutagenicity of AZT was further evaluated at the autosomal adenine phosphoribosyltransferase (APRT) gene. AZH1 cells, a human lymphoblastoid cell line heterozygous at the APRT locus, were exposed to 300 microM AZT for 0, 1, 3 or 6 days or to 0, 33, 100, 300 or 900 microM AZT for 3 days (n = 5 flasks/group). A cell cloning assay was used to quantitate APRT Mfs. AZT-induced APRT Mf increased with extended duration and with incremental concentrations of AZT exposure. There was a positive correlation (P = 0.022, coefficient = 0.93) between AZT incorporation into DNA and AZT-induced APRT Mfs. RFLP analyses indicated that AZT exclusively induced loss of heterozygosity in APRT mutants. These results, which are consistent with findings on the mutagenicity of AZT at the HPRT and TK genes, indicate the need for further investigations on the potential long-term side effects of AZT on humans, especially those who receive AZT for a prophylactic reason.

Relationships between DNA Incorporation, Mutant Frequency, and Loss of Heterozygosity at the TK Locus in Human Lymphoblastoid Cells Exposed to 3'-Azido-3'-deoxythymidine

3'-Azido-3'-deoxythymidine (AZT), a thymidine analogue widely used in the treatment of AIDS patients and for prevention of the onset of AIDS in HIV-seropositive individuals, causes tumors in mice exposed as adults or in utero. The purpose of this study was to investigate the potential mechanisms of AZT mutagenicity and carcinogenicity by quantifying the incorporation of AZT into cellular DNA, measuring AZT-induced thymidine kinase (TK) mutant frequencies (Mfs), and determining the percentage of loss of heterozygosity (LOH) in spontaneous or AZT-induced TK mutants in the human lymphoblastoid cell line, TK6. Cells were exposed to 300 microM AZT for 0, 1, 3, or 6 days, or to 0, 33, 100, 300, or 900 microM AZT for 3 days (n = 5 flasks/group). The effects of exposure concentration on incorporation of AZT into cellular DNA were evaluated by an AZT radioimmunoassay, and the effects of duration and concentration of AZT exposure on the TK Mfs were assessed by a cell-cloning assay. AZT was incorporated into DNA in a dose-related manner at concentrations up to 300 microM, above which no further increase was observed. TK Mf increased with the extended duration and with incremental concentrations of AZT exposure. There was a positive correlation (P = 0.036, coefficient = 0.903) between AZT-DNA incorporation and AZT-induced TK Mfs, suggesting that AZT incorporation into cellular DNA has a direct role in the genotoxicity of AZT. Southern blot analyses indicated that 84% (6.2 x 10(-6)/7.4 x 10(-6)) of AZT-induced mutants were attributable to LOH, consistent with the known mechanism of AZT as a DNA chain terminator. Considering the importance of LOH in human carcinogenesis, AZT-induced LOH warrants further study.

Genotoxicity of 3′-azido-3′-deoxythymidine in the human lymphoblastoid cell line, TK6: relationships between DNA incorporation, mutant frequency, and spectrum of deletion mutations in HPRT

Perinatal treatment with 3′-azido-3′-deoxythymidine (AZT) has been found to reduce the rate of maternal–infant transmission of HIV; however, AZT is genotoxic in mammalian cells in vitro and induces tumors in the offspring of mice treated in utero. The purpose of the present study was to investigate the relationships between incorporation of AZT into DNA, and the frequency and spectrum of mutations at the HPRT locus of the human lymphoblastoid cell line, TK6, following in vitro exposures to AZT. Cells were cultured in medium containing 0 or 300 μM AZT for 1, 3, or 6 day(s) ( n=5/group). The effects of exposure duration on incorporation of AZT into DNA and HPRT mutant frequency were determined using an AZT radioimmunoassay and a cell cloning assay, respectively. AZT accumulated in DNA in a supralinear manner, approaching a plateau at 6 days of treatment (101.9±14.7 molecules AZT/10 6 nucleotides). After 3 days of AZT exposure, HPRT mutant frequency was significantly increased (1.8-fold, p=0.016) compared to background (mutant frequency=3.78×10 −6). Multiplex PCR amplification of genomic DNA was used to determine the frequency of exon deletions in HPRT mutant clones from untreated cells versus AZT-treated cells. Molecular analyses of AZT-induced mutations revealed a significant difference in the frequency of total gene deletions (44/120 vs. 18/114 in controls, p=0.004 by the Mann–Whitney U-statistic). In fact, the Chi-square test of homogeneity demonstrate that the differences between the control and AZT-treatment groups is attributed mainly to this increase in total gene deletion mutations ( p=0.00001). These data indicate that the primary mechanism of AZT mutagenicity in human TK6 cells is through the production of large deletions which occur as a result of AZT incorporation into DNA and subsequent chain termination. The data imply that perinatal chemoprophylaxis with AZT may put children of HIV-infected women at potential risk for genetic damage.

Quantitative analysis of 3'-azido-3'-deoxythymidine incorporation into DNA in human colon tumor cells.

We have previously reported that 3'-azido-3'-deoxythymidine (AZT) can possess significant antineoplastic activity in vitro and in vivo when combined with agents which inhibit de novo thymidylate synthesis. Under these conditions cytotoxicity is closely associated with the degree to which AZT is incorporated into DNA. We now report a fluorescence postlabeling technique by which AZT incorporation into DNA can be quantitated without employing radiolabeled AZT. Cultured human colon tumor (HCT-8) cells were exposed to various concentrations of AZT alone and in combination with 5-fluorouracil (FUra). Control cells received the same amount of medium. DNA was isolated from harvested cell pellets (2 x 10(7)). Enzymatic digestion of DNA to the mononucleotide level followed by HPLC analysis of the digest showed that the DNA preparation was free of RNA contamination. The DNA digest was conjugated with dansyl chloride in situ via the phosphoramidate derivative with ethylenediamine. HPLC analysis of the postlabeled nucleotides using fluorescence detection detected 105, 245, and 479 fmol of 5'-monophosphate of AZT (AZTMP) per microg of DNA from cells exposed to 20, 50, and 100 microM AZT, respectively. FUra (3 microM) doubled the AZT incorporation per microg of DNA in cells exposed to 50 and 100 microM AZT. These findings generally support our previously reported data which quantitated (3H)AZT incorporation into cellular DNA and are discussed in light of the potential clinical utility of this technique in assessing the relationship between AZT incorporation into DNA and therapeutic action.

Cytotoxic and biochemical implications of combining AZT and AG-331.

We have reported that noncytotoxic concentrations of 3'-azido-3'-deoxythymidine (AZT) increase the cytotoxicity of ICI D1694, a folate-based thymidylate synthase (TS) inhibitor, with increasing AZT incorporation into DNA. We postulated that the inhibition of TS by ICI D1694 would decrease 5'-deoxythymidine triphosphate (dTTP) pools, which compete with AZT triphosphate (AZT-TP) as a substrate for DNA polymerase. Furthermore, the inhibition of TS would increase the activity of both thymidine kinase (TK) and thymidylate kinase (TdK). Each of these consequences of TS inhibition would favor more incorporation of AZT into DNA. Thus, we reasoned that other TS inhibitors should also result in increased AZT incorporation into DNA and, perhaps, in increased cytotoxicity. N6-[4-(Morpholinosulfonyl)benzyl]-N6-methyl-2,6-diaminobenz[ cd]indole glucuronate (AG-331) differs from ICI D1694 in that it is a de novo designed lipophilic TS inhibitor, it does not require a specific carrier for cellular uptake, and it does not undergo intracellular polyglutamation. This potent TS inhibitor causes minimal cytotoxicity in MGH-U1 human bladder cancer cells. A 24-h exposure to 5 microM AG-331 causes almost complete TS inhibition but only 35% cell kill. The combination of AZT and AG-331 in MGH-U1 cells resulted in an enhanced antitumor effect relative to that of each agent alone; 50 microM AZT, noncytotoxic alone, increased the cell kill of induced by AG-331 from 35% to 50%. Biochemical studies of this combination revealed that simultaneous treatment with 5 microM AG-331 plus 1.8 microM [3H]-AZT produced as much as a 68% +/- 7% increase in AZT incorporation into DNA. This observation was associated with an increase in DNA single-strand breaks, measured as comet tail moment, of up to 6.6-fold. These studies support our original premise that TS inhibition favors increased incorporation of AZT into DNA and that the combination causes more cell kill than either drug alone in MGH-U1 cells.

3′-azido-3′-deoxythymidine cytotoxicity and metabolism in the human colon tumor cell line HCT-8

We have reported that 3′-azido-3′-deoxythymidine (AZT) possesses significant cytotoxicity in human tumor models when combined with agents that inhibit de novo thymidylate (dTMP) synthesis, such as 5-fluorouracil (FUra) and methotrexate (MTX). To aid in the further development of these and related cancer chemotherapeutic regimens, this study was undertaken to identify the biochemical processes relevant to the induction of AZT cytotoxicity in the model human colon tumor cell line HCT-8. The ic 50 of AZT in this cell line after a 5-day exposure was 55 μM. In cells incubated for 5 days with various concentrations of [ 3H]AZT alone, both [ 3H]AZT nucleotide pools and [ 3H]AZT incorporation into DNA increased as the concentration of AZT in the medium increased. In addition, a 5-day exposure to AZT, at medium concentrations ⩽ 100 μM, resulted in a reduction in dTMP synthase (EC 2.1.1.45; methylene tetrahydrofolate:deoxyuridine-5′-monophosphate C methyltransferase) and dThd kinase (EC 2.7.1.27; ATP: thymidine phosphotransferase) activities, compared with cells incubated without drug. The ic 50 of AZT was unchanged when the medium concentration of dThd was increased from 0.1 to 50 μM. Increasing the concentration of dThd to 50 μM also did not affect intracellular pools of [ 3H]AZTDP and [ 3H]AZTTP or the degree to which [ 3H]AZT was incorporated into cellular DNA, but did reduce intracellular [ 3H]AZTMP by ∼ 75%. The degree to which 3′-amino-3′-deoxythymidine (AMT) was generated from AZT and incorporated into DNA also was not affected by varying the medium concentration of dThd. However, the amount of [ 3H]-AMT detected in DNA, ⩽ 3 pmol/10 6 cells at medium concentrations of [ 3H]AZT ⩽ 100 μM, was below that associated with significant cytotoxicity in these cells. These data support the notion that, in this model, AZT cytotoxicity is determined by the relative size of AZTTP pools and its utilization in DNA synthesis. Studies to verify this relationship assessed the effect of alterations in the concentration of dTTP and [ 3H]AZTTP on [ 3H]AZT incorporation into newly synthesized DNA in vitro, using DNA polymerases isolated from HCT-8 cells. The results of these studies confirmed that alterations in the concentration of either dTTP or AZTTP to reduce the dTTP/AZTTP ratio resulted in an increase in AZT incorporation into DNA. These findings are discussed in light of their biochemical implications and relevance to ongoing clinical trials.

Hydroxyurea enhances 3′‐azido‐3′‐deoxythymidine (AZT) cytotoxicity in human chronic myeloid leukemia models*

In this report we have evaluated the cytotoxic activity of 3'-azido-3'-deoxythymidine (AZT) used in combination with hydroxyurea (HU), an agent which disrupts de novo thymidylate synthesis. In 2 chronic myeloid leukemia (CML) cell lines, K562 and RWLeu4, the IC50 of AZT was 8 mumol/l and 28 mumol/l respectively, after a 5-day exposure, and the IC50 of HU was 80 mumol/l and 70 mumol/l respectively. In the presence of various concentrations of HU (1 mumol/l-100 mumol/l) the IC50 of AZT in both cell lines was significantly reduced and subsequent isobologram analysis revealed synergistic activity. Similarly, analysis of [3H]AZT incorporation into the DNA fraction of these cells indicated that exposure to AZT+HU resulted in an increased incorporation of AZT into DNA when compared to incubation in AZT alone. Biochemically, this effect appeared to be related to a decrease in dTTP pools caused by HU. The combination AZT+HU has also been demonstrated to exert a synergistic effect in inhibiting colony growth of bone marrow granulocyte-macrophage progenitors (CFU-GM) from patients affected by Ph1+ CML in chronic phase. These results are promising in view of a possible in vivo utilization of this drug combination.

Combination studies with 3′-azido-3′-deoxythymidine (AZT) plus ICI D1694: Cytotoxic and biochemical effects

The cytotoxicity of 3′-azido-3′-deoxythymidine (AZT), a thymidine analogue, and ICI D1694, a folate-based thymidylate synthase (TS) inhibitor, was examined individually and in combination in two human tumor cell lines, MGH-U1 bladder cancer and HCT-8 colon cancer cells, grown as a monolayer culture with and without thymidine (TdR). In addition, TS inhibition, [ 3H]AZT incorporation into DNA, [ 3H]AZT-MP (monophosphate) production, and DNA double-strand breaks were measured. Twenty-four hour exposure of AZT at 0.5, 5, 50 and 500 μM was not cytotoxic to MGH-U1 or HCT-8 cells in a colony-forming assay. ICI D1694 cytotoxicity increased with drug concentration, and the ic 50 and ic 90, respectively, were 0.0064 and 0.01 μM in MGH-U1 cells and 0.009 and 0.018 μM in HCT-8 cells. TdR in concentrations of 0.1 to 1.0 μM did not affect ICI D1694 cytotoxicity in either cell line. AZT at 5, 50 or 500 μM increased ICI D1694 cell kill. The ic 50 and ic 50 for MGH-U1 were 0.0037 and 0.0075 μM for 50 μM AZT combined with ICI D1694. The ic 90 for HCT-8 were 0.0075 and 0.015 μM for 50 μM AZT plus ICI D1694. The incorporation of [ 3H]AZT into DNA increased with increasing concentrations of ICI D1694. Concentrations producing an ic 50 and ic 90 of ICI D1694, respectively, increased incorporation of [ 3H]AZT into DNA by 319 and 569% in MHG-U1, and 243 and 400% in HCT-8 cells. The formation of [ 3H]AZT-MP paralleled the increase in [ 3H]AZT incorporated into DNA. AZT, ICI D1694 and the combination of AZT and ICI D1694 caused DNA double-strand breaks, with the combination of these agents being additive. CFU-GM survival, exposed to drug concentrations, as those used in the tumor cell lines, revealed that the therapeutic index was greater for AZT plus ICI D1694 than for ICI D1694 alone. These findings suggest that AZT plus ICI D1694 may increase antitumor effect with minimal myelosuppression. We conclude that AZT increases the cytotoxicity of ICI D1694 with increasing AZT incorporation into DNA.

Synergistic cytotoxicity of AZT plus alpha and gamma interferon in chronic myeloid leukemia cell line K562.

We have previously reported that the antineoplastic activity of 3'-azido 3' deoxythymidine (AZT) can be increased by drugs that inhibit "de novo" thymidylate synthesis, such as 5-fluorouracil, methotrexate and hydroxyurea. In the present study we tested the combinations AZT+alpha interferon (IFN) and AZT+gamma IFN on in vitro growth of the human acute-phase chronic myeloid leukemia (CML) cell line K562. After 72 hours incubation, not only AZT+alpha-IFN but also AZT+gamma-IFN were synergistic in inhibiting K562 growth, as demonstrated by isobologram analysis of the data. This enhanced cytotoxicity was confirmed by the evaluation of [3H]AZT incorporation into cellular DNA, that was increased by 50% and 222% in the presence of alpha- and gamma-IFN, respectively. The addition of 50 mumol/l thymidine to the culture medium was able to reduce the cytotoxicity of the drug combinations to the degree observed with each compound alone; furthermore, the increased incorporation of AZT into DNA was completely reversed. These data indicate the existence of a biochemical interaction between AZT and IFNs that results in an increased cytotoxic effect. While the combination AZT+alpha-IFN is currently being tested in HIV-related malignancies, AZT+gamma-IFN is new and deserves further study in human CML acute and chronic phase models, in view of possible clinical applications.

Incorporation of 3’-Azido-3’-deoxythymidine into Cellular DNA and Its Removal in a Human Leukemic Cell Line

3'-Azido-3'-deoxythymidine (AZT) is currently used in the treatment of patients with the acquired immunodeficiency syndrome (AIDS); this often, however, results in hematological toxicity. Although the mechanism of toxicity is not clear, it is thought to result in part from incorporation of AZT into DNA, which causes chain termination. In order to investigate the mechanism of AZT toxicity, the relationship between the presence of AZT in DNA of K562 cells, a chronic myelogenous leukemia cell line, and growth inhibition was examined. No growth inhibition was evident at less than 50 microM AZT, although incorporation of AZT into DNA was detected at 10 and 20 microM. This suggested that the presence of AZT in DNA was not sufficient to inhibit cell growth. Removal of AZT from the medium resulted in the removal of AZT from DNA of the cells, indicative of a cellular repair mechanism. Cellular DNA polymerases alpha, beta, gamma, and delta from human leukemic cells were inhibited by AZT trisphosphate to different degrees, polymerase alpha being the least potently inhibited. Furthermore, an enzyme with exonucleolytic activity, capable of removing AZT and dideoxycytidine from the correspondingly terminated DNA (in vitro), was obtained from these cells. In summary, AZT was incorporated into DNA at levels that were not toxic, and it could be removed by an exonuclease, which might play a key role in the susceptibility of cells to AZT.