Effects Of Azido-3'-deoxythymidine Research Articles

Differences in telomerase activity and the effects of AZT in aneuploid and euploid cells in colon cancer.

Telomerase-targeted treatments for cancer have received a great deal of attention because telomerase is detected in nearly all cancer cells but is not expressed in most normal tissues. Aneuploidy refers to a chromosome number that is not a multiple of the base chromosome number and can indicate either hypo- or hyperploid chromosome numbers. Most solid tumors are aneuploid. In the present study, we sought to determine whether there are differences in telomerase activity and hTERT gene expression between aneuploid and euploid cells. Furthermore, we investigated telomerase inhibitor 3'-azido-3'-deoxythymidine (AZT)-induced cell apoptosis using the p53-Puma/Noxa/Bax pathway and cell cycle arrest using the p53-p21 pathway in both aneuploid and euploid cells. Our results demonstrate that telomerase activity and hTERT gene expression were higher in aneuploid than in euploid cells. In addition, AZT exerted time- and dose-dependent cytotoxic effects on both aneuploid and euploid cells, and aneuploid cells were more sensitive to AZT-induced cytotoxicity. In addition, both the p53-Puma/Noxa/Bax pathway and the cell cycle arrest-associated p53-p21 pathway were involved in the AZT-induced suppression of tumor cells. Importantly, aneuploid cells were more sensitive to AZT-induced cell cycle arrest (p53-p21) and DNA double-strand breaks (γ-H2AX), while euploid cells were more sensitive to AZT-induced apoptosis (p53-Puma/Bax/Noxa).

Effects of AZT and RNA-protein complex (FA-2-b-β) extracted from Liang Jin mushroom on apoptosis of gastric cancer cells

To investigate the synergistic effects of 3'-azido-3'-deoxythymidine (AZT) and FA-2-b-beta extracted from Ling Jin mushroom on apoptosis of gastric cancer cells MKN45 in vitro. MTT analysis was made to examine the inhibition rate of MKN45 cells treated with AZT (2.5, 5, 10 and 20 mg/L) and FA-2-b-beta (5, 10, 20 and 40 mg/L) singly and combinatively for 24, 48 and 72 h. Apoptotic effects were evaluated by morphological methods, DNA agarose gel electrophoresis and flow cytometry, respectively. Telomerase activity was estimated by TRAP-ELISA. The mRNA expression of caspase-3 and Bcl-2 were detected by RT-PCR. AZT and FA-2-b-beta could significantly inhibit MKN45 cell proliferation and induce its apoptosis. MKN45 cells were inhibited in dose- and time- dependent manner. The inhibition effect of AZT combined with FA-2-b-beta was obviously better than that used singly (0.469 +/- 0.022 vs 1.075 +/- 0.055, P < 0.05, 0.325 +/- 0.029 vs 0.469 +/- 0.022 P < 0.01). AZT used singly and combination of FA-2-b-beta could decrease the activity of tumor cell telomerase, and AZT has synergistic function with FA-2-b-beta. A certain concentration of AZT could up-regulate the expression of caspase-3 mRNA (r = 0.9969, P < 0.01), which was positively related to apoptosis rate, and could down-regulate the expression of Bcl-2 mRNA, which was negatively related to apoptosis rate (r = 0.926, P < 0.01). Furthermore, the effect of AZT combined with FA-2-b-beta was significantly higher than that used singly. Combination of AZT and FA-2-b-beta has an obviously synergetic effect in the gastric cancer cells MKN45, which has provided a new approach to the treatment of gastric cancer clinically.

Site-Specific Reduction of Oxidative and Lipid Metabolism in Adipose Tissue of 3′-Azido-3′-Deoxythymidine-Treated Rats

Although it is well accepted that treatment with some nucleoside reverse transcriptase inhibitors modifies both fat metabolism and fat distribution in humans, the mechanisms underlying these modifications are not yet known. The present investigation examined whether a decrease in oxidative capacity, induced by a chronic oral administration of 3'-azido-3'-deoxythymidine (AZT) in rats, could be associated with an alteration of the lipogenic capacity of white adipose tissues. The impact of obesity as a factor was then evaluated. Results showed that AZT treatment induced differential effects depending on anatomical localization. Indeed, in the inguinal adipose tissue, the specific activities of cytochrome c oxidase and fatty acid synthase, two rate-controlling enzymes in energy and lipogenic metabolisms, respectively, both decreased under AZT treatment, thus leading to a lowered cell lipid accumulation. Moreover, the AMP-activated protein kinase phosphorylation level tended to increase, thus implying that AZT causes an energy imbalance. Furthermore, the inguinal tissue of obese rats presented a sensitivity to AZT treatment that was higher than that of lean rats. In contrast, for epididymal tissue, no significant change in all these parameters could be detected under AZT treatment, regardless of the nutritional status of the animals. Taken together, these data demonstrate differential effects of AZT on subcutaneous adipose tissue and visceral white adipose tissue. It could be considered that the chronic decreases in energy and lipogenic metabolism of inguinal adipocyte, consecutive to AZT treatment, may lead, in the long term, to adipose tissue atrophy.

Nature of DNA lesions induced in human hepatoma cells, human colonic cells and human embryonic lung fibroblasts by the antiretroviral drug 3′-azido-3′-deoxythymidine

This study tried to clarify the question if nuclear genotoxicity played a role in 3′-azido-3′-deoxythymidine (AZT) toxicity. We investigated cytotoxic and DNA-damaging effects of AZT on human hepatoma HepG2 and human colonic CaCo-2 cells as well as on human diploid lung fibroblasts HEL. The amount of induced DNA damage was measured by standard alkaline single cell gel electrophoresis (SCGE). The nature of induced DNA lesions was evaluated (1) by modified SCGE, which includes treatment of lysed cells with DNA repair enzymes Endo III and Fpg and enables to recognize oxidized bases of DNA, and (2) by SCGE processed in parallel at pH 13.0 (standard technique) and pH 12.1, which enables to recognize alkali labile DNA lesions and direct DNA strand breaks. Cytotoxicity of AZT was evaluated by the trypan blue exclusion technique. Our findings showed that 3-h treatment of cells with AZT decreased the viability of all cell lines studied. SCGE performed in the presence of DNA repair enzymes proved that AZT induced oxidative lesions to DNA in all cell types. In hepatoma HepG2 cells and embryonic lung fibroblasts HEL the majority of AZT-induced DNA strand breaks were pH-independent, i.e. they were identified at both pH values (12.1 and 13.0). These DNA lesions represented direct DNA breaks. In colonic Caco-2 cells DNA lesions were converted to DNA strand breaks particularly under strong alkaline conditions (pH > 13.0), which is characteristic for alkali-labile sites of DNA. DNA strand break rejoining was investigated by the standard comet assay technique during 48 h of post-AZT-treatment in HepG2 and Caco-2 cells. The kinetics of DNA rejoining, considered an indicator of DNA repair, revealed that AZT-induced DNA breaks were repaired in both cell types slowly, though HepG2 cells seemed to be more repair proficient with respect to AZT-induced DNA lesions.

Site specific alterations of adipose tissue mitochondria in 3′-azido-3′-deoxythymidine (AZT)-treated rats: An early stage in lipodystrophy?

Although it is well accepted that treatment with nucleoside reverse transcriptase inhibitors (NRTIs) modifies fat metabolism and fat distribution in humans, the mechanisms underlying these modifications are not yet known. The present investigation examines the effects of chronic oral administration of 3′-azido-3′-deoxythymidine (AZT) on the mitochondrial metabolism and the redox status management of rat white adipose tissues originating from two anatomical sites, as well as of the rat liver. Results showed that AZT treatment induced differential effects on the mitochondrial functions depending on the anatomical localisation. Indeed, in inguinal adipose tissue, a significant decrease in the cytochrome c oxidase activity and in the mitochondrial DNA (mtDNA) content was observed, whereas the activity of citrate synthase, a mitochondrial protein exclusively encoded by the nucleus, was not affected. In contrast, no significant change in these parameters could be detected for epididymal tissue and for liver. In parallel, no oxidative stress could be detected after treatment, for both white adipose tissues and for liver, even though treated liver exhibited several modifications in redox management. Taken together, these data demonstrate differential mitochondrial effects of AZT on subcutaneous versus visceral white adipose tissue. Moreover, the decrease in mitochondrial oxidative capacity of inguinal adipocyte consecutive to AZT treatment is not primarily due to an oxidative stress per se, but rather to a depletion of the mtDNA content per cell.

Effects of AZT on cellular iron homeostasis.

3'-azido-3'-deoxythymidine (AZT), the first chemotherapeutic drug approved by FDA for treatment of HIV-infected patients and still used in combination therapy, has been shown to induce, upon prolonged exposure, severe bone marrow toxicity manifested as anemia, neutropenia and siderosis. These toxic effects are caused by inhibition of heme synthesis and, as a consequence, transferrin receptor (TfR) number appears increased and so iron taken up by cells. Since iron overload can promote the frequency and severity of many infections, siderosis is viewed as a further burden for AIDS patients. We have previously demonstrated that AZT-treated K562 cells showed an increase of the number of TfRs located on the surface of the plasma membrane without affecting their biosynthesis, but slowing down their endocytotic pathway. In spite of the higher number of receptors on the plasma-membrane of AZT-treated cells, intracellular accumulation of iron showed a similar level in control and in drug-exposed cells. The chelating ability of AZT and of its phosphorylated derivatives, both in an acellular system and in K562 cells, was also checked. The results demonstrated that AZT and AZTMP were uneffective as iron chelators, while AZTTP displayed a significant capacity to remove iron from transferrin (Tf). Our results suggest that AZT may be not directly involved in the iron overloading observed upon its prolonged use in AIDS therapy. The iron accumulation found in these patients is instead caused by other unknown mechanisms that need further studies to be clarified.

3′-Azido-3′-deoxythymidine (AZT) induces apoptosis and alters metabolic enzyme activity in human placenta

The anti-HIV drug 3′-azido-3′-deoxythymidine (AZT) is the drug of choice for preventing maternal–fetal HIV transmission during pregnancy. Our aim was to assess the cytotoxic effects of AZT on human placenta in vitro. The mechanisms of AZT-induced effects were investigated using JEG-3 choriocarcinoma cells and primary explant cultures from term and first-trimester human placentas. Cytotoxicity measures included trypan blue exclusion, MTT, and reactive oxygen species (ROS) assays. Apoptosis was measured with an antibody specific to cleaved caspase-3 and by rescue of cells by the general caspase inhibitor Boc-D-FMK. The effect of AZT on the activities of glutathione- S-transferase, β-glucuronidase, UDP-glucuronosyl transferase, cytochrome P450 (CYP) 1A, and CYP reductase (CYPR) in the placenta was assessed using biochemical assays and immunoblotting. AZT increased ROS levels, decreased cellular proliferation rates, was toxic to mitochondria, and initiated cell death by a caspase-dependent mechanism in the human placenta in vitro. In the absence of serum, the effects of AZT were amplified in all the models used. AZT also increased the amounts of activity of GST, β-glucuronidase, and CYP1A, whereas UGT and CYPR were decreased. We conclude that AZT causes apoptosis in the placenta and alters metabolizing enzymes in human placental cells. These findings have implications for the safe administration of AZT in pregnancy with respect to the maintenance of integrity of the maternal–fetal barrier.

3′-Azidothymidine Potently Inhibits the Biosynthesis of Highly Branched N-Linked Oligosaccharides and Poly-N-acetyllactosamine Chains in Cells

3′-Azidothymidine Potently Inhibits the Biosynthesis of Highly Branched N-Linked Oligosaccharides and Poly-N-acetyllactosamine Chains in Cells

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.

The M184V mutation in the reverse transcriptase of human immunodeficiency virus type 1 impairs rescue of chain-terminated DNA synthesis.

Nucleoside analog chain terminators such as 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxy-3'-thiacytidine (3TC) represent an important class of drugs that are used in the clinic to inhibit the reverse transcriptase (RT) of human immunodeficiency virus type 1. Recent data have suggested that mutant enzymes associated with AZT resistance are capable of removing the chain-terminating residue with much greater efficiency than wild-type RT and this may, in turn, facilitate rescue of DNA synthesis; these experiments were performed using physiological concentrations of pyrophosphate or nucleoside triphosphates, respectively. The present study demonstrates that the M184V mutation, which confers high-level resistance to 3TC, can severely compromise the removal of chain-terminating nucleotides. Pyrophosphorolysis on 3TC-terminated primer strands was not detectable with M184V-containing, as opposed to wild-type, RT, and rescue of AZT-terminated DNA synthesis was significantly decreased with the former enzyme. Thus, mutated RTs associated with resistance to AZT and 3TC possess opposing, and therefore incompatible, phenotypes in this regard. These results are consistent with tissue culture and clinical data showing sustained antiviral effects of AZT in the context of viruses that contain the M184V mutation in the RT-encoding gene.

Long-term effects of prenatal 3'-azido-3'-deoxythymidine (AZT) exposure on intermale aggressive behaviour of mice.

AZT treatment of seropositive pregnant women and their neonates has been widely used due to its effectiveness in reducing vertical transmission of HIV, but medium- and long-term effects of AZT on neurobehavioural development and adult responding are still poorly described. The aim of the present study was to evaluate the long-term effects of prenatal AZT treatment on aggressive behaviour of adult male mice. Pregnant CD-1 mice were given saline vehicle, 0.4, or 0.8 mg/ml AZT in their drinking water from gestation day 10 to delivery. Social-aggressive types of interaction were assessed in their male offspring following a 4-week isolation period. Two groups of subjects were used, each undergoing a different type of test: test 1 consisted of a single 20-min encounter with an isolated same-strain opponent on postnatal day (PND) 90, while in test 2 (PND 150) subjects were paired for 10 min for 5 consecutive days with a non-isolated opponent. Slight changes in both aggressive and defensive components of the male-specific agonistic pattern were evident only in test 1, AZT mice displaying a limited increase of aggressive behaviour compared to their controls. Although the long-term effects of prenatal AZT on social behaviour are limited, they may be of some relevance for paediatricians in order to plan a follow-up of infants, children and adolescents exposed in utero to antiretroviral drugs.

The antiretrovirus drug 3'-azido-3'-deoxythymidine increases the retrovirus mutation rate.

It was previously observed that the nucleoside analog 5-azacytidine increased the spleen necrosis virus (SNV) mutation rate 13-fold in one cycle of retrovirus replication (V. K. Pathak and H. M. Temin, J. Virol. 66:3093-3100, 1992). Based on this observation, we hypothesized that nucleoside analogs used as antiviral drugs may also increase retrovirus mutation rates. We sought to determine if 3'-azido-3'-deoxythymidine (AZT), the primary treatment for human immunodeficiency virus type 1 (HIV-1) infection, increases the retrovirus mutation rate. Two assays were used to determine the effects of AZT on retrovirus mutation rates. The strategy of the first assay involved measuring the in vivo rate of inactivation of the lacZ gene in one replication cycle of SNV- and murine leukemia virus-based retroviral vectors. We observed 7- and 10-fold increases in the SNV mutant frequency following treatment of target cells with 0.1 and 0.5 microM AZT, respectively. The murine leukemia virus mutant frequency increased two- and threefold following treatment of target cells with 0.5 and 1.0 microM AZT, respectively. The second assay used an SNV-based shuttle vector containing the lacZ alpha gene. Proviruses were recovered as plasmids in Escherichia coli, and the rate of inactivation of lacZ alpha was measured. The results indicated that treatment of target cells increased the overall mutation rate two- to threefold. DNA sequence analysis of mutant proviruses indicated that AZT increased both the deletion and substitution rates. These results suggest that AZT treatment of HIV-1 infection may increase the degree of viral variation and alter virus evolution or pathogenesis.

Effect of recombinant human hemoglobin on human bone marrow progenitor cells: protection and reversal of 3′ -azido-3′ -deoxythymidine-induced toxicity

Long-term therapy of AIDS patients with 3′ -azido-3′ -deoxythymidine (AZT) is limited by hematopoietic toxicity. While the mechanism(s) of this toxicity remain elusive, various strategies are being developed to reduce these toxic effects including combination therapy with non-myelotoxic anti-human immunodeficiency virus (HIV) drugs and/or administration of protective or rescue agents, such as cytokines and growth factors. Using a physiologically relevant human CD34+ bone marrow cell liquid culture system, a crosslinked human recombinant hemoglobin (rHb), currently in Phase II clinical trials, was investigated for effects on hematopoiesis and for its potential in protecting or reversing AZT-induced hematopoietic toxicity. These investigations demonstrated that 0.01, 0.1, or 1 μM human rHb did not affect the proliferation of erythroid or myeloid lineage cells. A concentration of 1 μM rHb partially protected erythroid lineage cells from an inhibition of proliferation induced by 0.1 and 1 μM AZT. Inhibition of proliferation of cells previously exposed to AZT was not reversed at this concentration. These data suggest that human rHb may be of benefit in reducing the toxic effects of AZT in the bone marrow of AIDS patients.

Intracellular Metabolism of 3′-Azido-3′-Deoxythymidine in the Presence of Ganciclovir or Foscarnet

Comparisons were made between the intracellular phosphorylation of 3′-Azido-3′-deoxythymidine (AZT) alone and in combination with ganciclovir (GCV) or foscarnet (PFA) in lymphocytes, uninfected fibroblasts and CMV-infected fibroblasts. The effects of AZT and the combinations of AZT with GCV or PFA on cellular dNTP pools were also examined. The phosphorylation of AZT was not altered by the presence of GCV or PFA in lymphocytes, and neither AZT nor the combinations of AZT with GCV or PFA changed the levels of cellular dNTP pools in these cells. AZT was phosphorylated to a greater extent in lymphocytes when compared to fibroblasts, but the proportion of AZT di- and triphosphates was greater in fibroblasts. The infection of fibroblasts with CMV enhanced AZT phosphorylation and increased the levels of cellular dNTP pools. GCV caused a specific reduction in AZT phosphorylation in CMV-infected fibroblasts, with a seven-fold drop in AZT triphosphate compared to AZT alone. GCV did not affect AZT phosphorylation in uninfected fibroblasts, nor did GCV reduce the dTTP pool compared to AZT alone. The effects of GCV upon AZT phosphorylation in CMV-infected cells may shed light on the antagonistic effects of GCV upon the anti-HIV activity of AZT, and are of importance for the development of effective combination therapies for the treatment of AIDS patients infected with CMV.

3'-Azido-3'-deoxythymidine inhibits erythroid-specific transcription factors in human erythroid K562 leukemia cells.

The present study examines genetic mechanism(s) possibly involved in the observed 3'-azido-3'-deoxythymidine (AZT)-induced inhibition of globin gene transcription by evaluating the direct phenotypic erythroid effects of AZT on erythroid-specific transcription factors which regulate globin gene promoters. In vitro binding of GATA-1 or NFE-2 to its consensus sequence was decreased in the presence of AZT reaching a maximum inhibition as early as 24 h after AZT treatment. Nuclear extracts from butyric acid-induced K562 cells treated with an IC50 concentration of AZT exhibited a decrease in GATA-1 and NFE-2 binding by approximately 30% and 35%. In contrast, 2',3'-dideoxycytidine which inhibits cell growth without affecting hemoglobin synthesis, had no effect on binding of GATA-1 and NFE-2 factors. Northern blot analysis revealed a 25% decrease by AZT in GATA-1 mRNA steady-state levels at 24 h and this inhibitory effect was maintained until 72 h after drug addition. A similar decrease in NFE-2 mRNA steady-state levels was observed at 72 h after AZT treatment. This study suggests that AZT inhibition of erythroid differentiation is subsequent to a decrease of nuclear factors gene expression which affect their DNA binding.

In vitro effects of 3'-azido-3'-deoxythymidine (AZT) on normal human polymorphonuclear cell and monocyte-macrophage functional capacities.

The in vitro effects of 3'-azido-3'-deoxythymidine (AZT) (at concentrations of 1, 10 and 100 microM, respectively) on normal human polymorphonuclear cell (PMN) and monocyte-macrophage functional capacities were evaluated. Results show that AZT was able to decrease monocyte phagocytosis only, while PMN polarization, phagocytosis and killing were unaffected by drug pretreatment. Quite interestingly, monocyte-derived macrophages maintained their unaltered phagocytic function in spite of the presence of AZT in overnight cultures, thus indicating that monocytes are more susceptible than macrophages to the antiproliferative effects of AZT. Since our data indicate that AZT affects normal human monocyte phagocytosis, it is advisable to evaluate this immune parameter in HIV+ patients administered with this drug.

DNA found in human immunodeficiency virus type 1 particles may not be required for infectivity.

We have studied the presence and significance of retroviral genome-derived DNA in the core of human immunodeficiency virus (HIV) particles produced from transfections of HXB2 expression vectors in COS-7 cells and from HIV type 1 IIIB chronically infected H9 cells. Viruses purified by sucrose cushion centrifugation and treated with DNase I contained 1000-fold more viral RNA than DNA. However, protease-defective viruses that contained only p160gag-pol had less than 100 times the amount of DNA in their cores than wild-type viruses suggesting that the p66/p51 form of reverse transcriptase was responsible for DNA transcription. Viruses produced by transfections in the presence of 3'-azido-3'-deoxythymidine (AZT) contained the viral RNA genome but only DNA of premature length because of the chain terminating effects of AZT. However such viruses were as infections for CD4+ cells as wild-type virus. We conclude that retrovirus-derived DNA in HIV-1 particles is not required for infection and does not play a significant role in this process.

Inhibition of human immunodeficiency virus type 1 integrase by 3'-azido-3'-deoxythymidylate.

The effects of 3'-azido-3'-deoxythymidine (AZT) and three of its intracellular metabolites, azido- thymidine mono-, di-, and triphosphates, on the human immunodeficiency virus type 1 integrase have been determined. AZT mono-, di-, and triphosphate have an IC50 for integration between 110 and 150 microM, whereas AZT does not inhibit the integrase. The inhibition by AZT monophosphate can be partially reversed by coincubation with either thymidine monophosphate or 2',3'-dideoxythymidine monophosphate, suggesting that either of these monophosphates can bind to the integrase but that the azido group at the 3' position could be responsible for the inhibition. Integrase inhibition is associated with reduced enzyme-DNA binding but does not appear to be competitive with respect to the DNA substrate. Inhibition of an integrase deletion mutant containing only amino acids 50-212 suggests that these nucleotides bind in the catalytic core. Concentrations up to 1 mM AZT monophosphate can accumulate in vivo, indicating that integrase inhibition may contribute to the antiviral effects of AZT. The increasing incidence of AZT-resistant virus strains may, therefore, be associated with mutations not only in the reverse transcriptase but also in the human immunodeficiency virus integrase. Finally, these observations suggest that additional strategies for antiviral drug development could be based upon nucleotide analogs as inhibitors of human immunodeficiency virus integrase.

The effects of AZT and DDI on pre- and postimplantation mammalian embryos: an in vivo and in vitro study.

This study reports the effects of the nucleoside analogs dideoxyinosine (DDI) and 3'-azido-3'-deoxythymidine (AZT) on mammalian embryonic development. When administered to pregnant mice (at concentrations ranging from 10 to 300 mg/kg/day), through all or part of gestation, AZT and DDI did not result in any visible effect on mouse embryos nor did they cause any obvious malformation or defect at birth or during postnatal growth. Similarly, when embryonic or fetal mouse or human cells (from brain, limb buds, or different organ rudiments) were exposed to AZT or DDI in vitro, cytotoxicity was observed only in the mM range, with AZT showing slightly higher cytotoxicity and brain cells appearing slightly more sensitive to both nucleosides. However, even in cultures treated with very high concentrations of AZT or DDI, the reduction in the number of terminally differentiated skeletal myotubes, cardiocytes, neurons, and chondrocytes was similar to the reduction in the total number of cells, indicating that AZT and DDI did not selectively inhibit differentiation of any of the above-mentioned cell types. Finally, preimplantation mouse embryos (at the 2-cell or 4-cell stage), treated in vitro with micromolar concentrations of AZT, were arrested at the 4-cell stage. DDI or other nucleoside analogs tested did not have this effect.

Acute effects of 3′-azido-3′-deoxythymidine on the cell cycle of HL60 cells

The average doubling times of HL60 cells grown in the presence of 0,5,10 and 100 microM 3'-azido-3'-deoxythymidine (AZT) for 72 h were, respectively, 51.1, 65.7, 69.0 and 76.3 h. This drug-concentration-dependent prolongation of the cell-doubling time was associated with a progressive increase in modal cell volume. The technique of combined Feulgen microspectrophotometry and 3H-thymidine autoradiography revealed that the cell cycle distribution of HL60 cells cultured in the presence of 5 and 100 microM AZT for 48 h was abnormal, with an increased percentage of cells in the S phase and a decreased percentage in the G1phase. From the cell doubling times and the cell cycle distribution data, and making a number of assumptions, upper limit estimates for the duration of the S phase in cultures containing 0,5 and 100 microM AZT were calculated to be 26.2, 35.8 and 49.6 h, respectively. The data indicate that concentrations of AZT achieved in the plasma of patients receiving this drug (i.e. 5 microM) cause a substantial prolongation of both the cell cycle time and the duration of the S phase of HL60 cells. It therefore seems likely that some of the toxic effects of AZT seen in vivo, including impairment of bone marrow function, are at least partly related to AZT-induced disturbances of DNA synthesis and proliferation in human cells.