Chemical interference with DSIF complex formation lowers synthesis of mutant huntingtin gene products and curtails mutant phenotypes.

Earlier work has shown that siRNA-mediated reduction of the SUPT4H or SUPT5H proteins, which interact to form the DSIF complex and facilitate transcript elongation by RNA polymerase II (RNAPII), can decrease expression of mutant gene alleles containing nucleotide repeat expansions differentially. Using luminescence and fluorescence assays, we identified chemical compounds that interfere with the SUPT4H-SUPT5H interaction and then investigated their effects on synthesis of mRNA and protein encoded by mutant alleles containing repeat expansions in the huntingtin gene (HTT), which causes the inherited neurodegenerative disorder, Huntington's Disease (HD). Here we report that such chemical interference can differentially affect expression of HTT mutant alleles, and that a prototypical chemical, 6-azauridine (6-AZA), that targets the SUPT4H-SUPT5H interaction can modify the biological response to mutant HTT gene expression. Selective and dose-dependent effects of 6-AZA on expression of HTT alleles containing nucleotide repeat expansions were seen in multiple types of cells cultured in vitro, and in a Drosophila melanogaster animal model for HD. Lowering of mutant HD protein and mitigation of the Drosophila "rough eye" phenotype associated with degeneration of photoreceptor neurons in vivo were observed. Our findings indicate that chemical interference with DSIF complex formation can decrease biochemical and phenotypic effects of nucleotide repeat expansions.

6-Azauridine Induces Autophagy-Mediated Cell Death via a p53- and AMPK-Dependent Pathway.

6-Azauridine (6-AZA), a pyrimidine nucleoside analogue, is known to exhibit both antitumor and antiviral activities. Although 6-AZA was discovered more than 60 years ago, the cellular effects of this compound are yet to be elucidated. Here, we report that 6-AZA regulates autophagy-mediated cell death in various human cancer cells, where 6-AZA treatment activates autophagic flux through the activation of lysosomal function. Furthermore, 6-AZA exhibited cytotoxicity in all cancer cells studied, although the mechanisms of action were diverse. In H460 cells, 6-AZA treatment induced apoptosis, and the extent of the latter could be reduced by treatment with chloroquine (CQ), a lysosomal inhibitor. However, 6-AZA treatment resulted in cell cycle arrest in H1299 cells, which could not be reversed by CQ. The cytotoxicity associated with 6-AZA treatment could be linearly correlated to the degree of autophagy-mediated cell death. In addition, we demonstrated that the cytotoxic effect of 6-AZA was dependent on AMPK and p53. These results collectively indicate that autophagy-mediated cell death triggered by 6-AZA contributes to its antitumor effect.

Identification of Inhibitors of ZIKV Replication.

Zika virus (ZIKV) was identified in 1947 in the Zika forest of Uganda and it has emerged recently as a global health threat, with recurring outbreaks and its associations with congenital microcephaly through maternal fetal transmission and Guillain-Barré syndrome. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or antivirals to treat ZIKV infections, which underscores an urgent medical need for the development of disease intervention strategies to treat ZIKV infection and associated disease. Drug repurposing offers various advantages over developing an entirely new drug by significantly reducing the timeline and resources required to advance a candidate antiviral into the clinic. Screening the ReFRAME library, we identified ten compounds with antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV). Moreover, we showed the ability of these ten compounds to inhibit influenza A and B virus infections, supporting their broad-spectrum antiviral activity. In this study, we further evaluated the broad-spectrum antiviral activity of the ten identified compounds by testing their activity against ZIKV. Among the ten compounds, Azaribine (SI-MTT = 146.29), AVN-944 (SI-MTT = 278.16), and Brequinar (SI-MTT = 157.42) showed potent anti-ZIKV activity in post-treatment therapeutic conditions. We also observed potent anti-ZIKV activity for Mycophenolate mofetil (SI-MTT = 20.51), Mycophenolic acid (SI-MTT = 36.33), and AVN-944 (SI-MTT = 24.51) in pre-treatment prophylactic conditions and potent co-treatment inhibitory activity for Obatoclax (SI-MTT = 60.58), Azaribine (SI-MTT = 91.51), and Mycophenolate mofetil (SI-MTT = 73.26) in co-treatment conditions. Importantly, the inhibitory effect of these compounds was strain independent, as they similarly inhibited ZIKV strains from both African and Asian/American lineages. Our results support the broad-spectrum antiviral activity of these ten compounds and suggest their use for the development of antiviral treatment options of ZIKV infection.

Identification and evaluation of antivirals for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is the causative agent of Rift Valley fever (RVF) that affects both livestock and humans. There are neither fully licensed RVF vaccines available for human or animal use, nor effective antiviral drugs approved for human use in the U.S. To identify antiviral compounds effective for RVF, we developed and employed a cell-based high-throughput assay using a recombinant RVFV MP-12 strain, which expresses Renilla luciferase in place of the NSs protein, to screen 727 small compounds purchased from the National Institutes of Health. Twenty-three compounds were initially identified using the screening assay. Two compounds, 6-azauridine and mitoxantrone, also inhibited the replication of the parental MP-12 strain encoding the NSs gene, with limited cytotoxic effects. The respective 50% inhibitory concentrations were 29.07 µM and 79.85 µM when tested with the parental MP-12 strain at a multiplicity of infection of 2. The compounds were further evaluated using the STAT-1 KO mouse model. At one hour post intranasal inoculation of MP-12 strain, mice were intranasally treated with each indicated compound twice daily. Mice treated with either placebo or 6-azauridine displayed severe weight loss and reached the threshold for euthanasia with obvious neurologic symptoms. Onset of disease was, however, delayed in mice treated with either ribavirin or mitoxantrone. The results indicated that mitoxantrone can reduce the severity of diseases in RVFV-infected mice. Our studies build the foundation for the initial screening and efficacy studies of RVF antivirals in a BSL-2 environment, avoiding the higher risks of BSL-3 exposure with wild-type virus.

Novel 5-substituted derivatives of 2'-deoxy-6-azauridine with antibacterial activity.

The emergence of new drug-resistant strains of bacteria necessitates the development of principally new antibacterial agents. One of the novel classes of antibacterial agents is nucleoside analogs. We have developed a fast and simple one-pot method for preparation of α- and ß-anomers of 5-modified 6-aza- and 2-thio-6-aza-2'-deoxyuridine derivatives in high yields. 2-Thio derivatives demonstrated moderate activity against Mycobacterium smegmatis (MIC = 0.2-0.8 mM), Staphylococcus aureus (MIC = 0.03-0.9 mM) and some other Gram-positive bacteria. 2'-Deoxy-2-thio-5-phenyl-6-azauridine (2b) effectively suppressed the growth of Gram-negative bacteria Pseudomonas aeruginosa ATCC 27853 (MIC = 0.03 mM)-the one that causes diseases difficult to treat due to high resistance to antibiotics. 5'-Monophosphates of compounds 2a, b and 3a, b were docked into a binding site of Mycobacterium tuberculosis flavin-dependent thymidylate synthase (ThyX) enzyme. The molecular modeling demonstrates the possibility of binding of the 5-modified 2-thio-6-aza-2'-deoxyuridine 5'-monophosphates within the active site of the enzyme and thereby inhibiting the growth of the bacteria.

Synthesis of New Potential Lipophilic Co-Drugs of 2-Chloro-2'-deoxyadenosine (Cladribine, 2-CdA, Mavenclad®, Leustatin®) and 6-Azauridine (z6 U) with Valproic Acid.

2-Chloro-2'-deoxyadenosine (cladribine, 1) was acylated with valproic acid (2) under various reaction conditions yielding 2-chloro-2'-deoxy-3',5'-O-divalproyladenosine (3) as well as the 3'-O- and 5'-O-monovalproylated derivatives, 2-chloro-2'-deoxy-3'-O-valproyladenosine (4) and 2-chloro-2'-deoxy-5'-O-valproyladenosine (5), as new co-drugs. In addition, 6-azauridine-2',3'-O-(ethyl levulinate) (8) was valproylated at the 5'-OH group (→9). All products were characterized by 1 H- and 13 C-NMR spectroscopy and ESI mass spectrometry. The structure of the by-product 6 (N-cyclohexyl-N-(cyclohexylcarbamoyl)-2-propylpentanamide), formed upon valproylation of cladribine in the presence of N,N-dimethylaminopyridine and dicyclohexylcarbodiimide, was analyzed by X-ray crystallography. Cladribine as well as its valproylated co-drugs were tested upon their cancerostatic/cancerotoxic activity in human astrocytoma/oligodendroglioma GOS-3 cells, in rat malignant neuro ectodermal BT4Ca cells, as well as in phorbol-12-myristate 13-acetate (PMA)-differentiated human THP-1 macrophages. The most important result of these experiments is the finding that only the 3'-O-valproylated derivative 4 exhibits a significant antitumor activity while the 5'-O- as well as the 3',5'-O-divalproylated cladribine derivatives 3 and 5 proved to be inactive.

Comparison of three dimensional synergistic analyses of percentage versus logarithmic data in antiviral studies.

Cell culture antiviral experiments were conducted in order to understand the relationship between percentage data generated by plaque reduction (PR) and logarithmic data derived by virus yield reduction (VYR) assays, using three-dimensional MacSynergy II software. The relationship between percentage and logarithmic data has not been investigated previously. Interpretation of drug-drug interactions is based on a Volume of Synergy (VS) calculation, which can be positive (synergy), negative (antagonistic), or neutral (no or minimal interaction). Interactions of two known inhibitors of vaccinia virus replication, cidofovir and 6-azauridine, used in combination by PR assay yielded a VS value of 265, indicative of strong synergy. By VYR, the VS value was only 37, or weak synergy using the same criterion, even though profound log10 reductions in virus titer occurred at multiple drug combinations. These results confirm that the differences in VS values is dependent of the measurement scale, and not that the degree of synergy differed between the assays. We propose that for logarithmic data, the calculated VS values will be lower for significant synergy and antagonism and that volumes of >10 µM2log10 PFU/ml (or other units such as µM2log10 genomic equivalents/ml or µM2log10 copies/ml) and <-10 µM2log10 PFU/ml are likely to be indicative of strong synergy and strong antagonism, respectively. Data presented here show that the interaction of cidofovir and 6-azauridine was strongly synergistic in vitro.

Sesquiterpenoids with Various Carbocyclic Skeletons from the Flowers of Chrysanthemum indicum.

A phytochemical investigation of the flowers of Chrysanthemum indicum yielded sesquiterpenoids 1-25 with various carbocyclic skeletons, including 10 new (1-10) and 15 known (11-25) analogues. The structures were elucidated via their physical data, while the absolute configuration of compounds 6, 8, and 10 was assessed via electronic circular dichroism analysis. The evaluation of the effect of sesquiterpenoids on porcine epidemic diarrhea virus (PEDV) replication showed that compounds 1-5, 12, 14, 16, 17, 19, and 21 increased cell viability against cell death in PEDV-injected cells. Compounds 2, 12, and 17 were selected and investigated for their inhibition of proteins required for PEDV replication. Compounds 2 and 17 significantly reduced PEDV nucleocapsid and spike protein synthesis compared with azauridin as a positive control.

Evaluation of anti-Zika virus activities of broad-spectrum antivirals and NIH clinical collection compounds using a cell-based, high-throughput screen assay.

Recent studies have clearly underscored the association between Zika virus (ZIKV) and severe neurological diseases such as microcephaly and Guillain-Barre syndrome. Given the historical complacency surrounding this virus, however, no significant antiviral screenings have been performed to specifically target ZIKV. As a result, there is an urgent need for a validated screening method and strategy that is focused on highlighting potential anti-ZIKV inhibitors that can be further advanced via rigorous validation and optimization. To address this critical gap, we sought to test whether a cell-based assay that measures protection from the ZIKV-induced cytopathic effect could serve as a high-throughput screen assay for discovering novel anti-ZIKV inhibitors. Employing this approach, we tested the anti-ZIKV activity of previously known broad-spectrum antiviral compounds and discovered several compounds (e.g., NITD008, SaliPhe, and CID 91632869) with anti-ZIKV activity. Interestingly, while GTP synthesis inhibitors (e.g., ribavirin or mycophenolic acid) were too toxic or showed no anti-ZIKV activity (EC50 > 50 µM), ZIKV was highly susceptible to pyrimidine synthesis inhibitors (e.g., brequinar) in the assay. We amended the assay into a high-throughput screen (HTS)-compatible 384-well format and then screened the NIH Clinical Compound Collection library, which includes a total of 727 compounds organized, using an 8-point dose response format with two Zika virus strains (MR766 and PRVABC59, a recent human isolate). The screen discovered 6-azauridine and finasteride as potential anti-ZIKV inhibitors with EC50 levels of 3.18 and 9.85 µM for MR766, respectively. We further characterized the anti-ZIKV activity of 6-azauridine and several pyrimidine synthesis inhibitors such as brequinar in various secondary assays including an antiviral spectrum test within flaviviruses and alphaviruses, Western blot (protein), real-time PCR (RNA), and plaque reduction assays (progeny virus). From these assays, we discovered that brequinar has potent anti-ZIKV activity. Our results show that a broad anti-ZIKV screen of compound libraries with our CPE-based HTS assay will reveal multiple chemotypes that could be pursued as lead compounds for therapies to treat ZIKV-associated diseases or as molecular probes to study the biology of the ZIKV replication mechanism.

Lassa and Ebola virus inhibitors identified using minigenome and recombinant virus reporter systems.

Lassa virus (LASV) and Ebola virus (EBOV) infections are important global health issues resulting in significant morbidity and mortality. While several promising drug and vaccine trials for EBOV are ongoing, options for LASV infection are currently limited to ribavirin treatment. A major factor impeding the development of antiviral compounds to treat these infections is the need to manipulate the virus under BSL-4 containment, limiting research to a few institutes worldwide. Here we describe the development of a novel LASV minigenome assay based on the ambisense LASV S segment genome, with authentic terminal untranslated regions flanking a ZsGreen (ZsG) fluorescent reporter protein and a Gaussia princeps luciferase (gLuc) reporter gene. This assay, along with a similar previously established EBOV minigenome, was optimized for high-throughput screening (HTS) of potential antiviral compounds under BSL-2 containment. In addition, we rescued a recombinant LASV expressing ZsG, which, in conjunction with a recombinant EBOV reporter virus, was used to confirm any potential antiviral hits in vitro. Combining an initial screen to identify potential antiviral compounds at BSL-2 containment before progressing to HTS with infectious virus will reduce the amount of expensive and technically challenging BSL-4 containment research. Using these assays, we identified 6-azauridine as having anti-LASV activity, and demonstrated its anti-EBOV activity in human cells. We further identified 2'-deoxy-2'-fluorocytidine as having potent anti-LASV activity, with an EC50 value 10 times lower than that of ribavirin.

2-thio-6-azauridine inhibits Vpu mediated BST-2 degradation.

BACKGROUD: BST-2 is an interferon-induced host restriction factor that inhibits the release of diverse mammalian enveloped viruses from infected cells by physically trapping the newly formed virions onto the host cell surface. Human Immunodeficiency Virus-1 (HIV-1) encodes an accessory protein Vpu that antagonizes BST-2 by down-regulating BST-2 from the cell surface. RESULTS: Using a cell-based ELISA screening system, we have discovered a lead compound, 2-thio-6-azauridine, that restores cell surface BST-2 level in the presence of Vpu. This compound has no effect on the expression of BST-2 and Vpu, but inhibits Vpu-mediated BST-2 down-regulation and exerts no effect on Vpu-induced down-regulation of CD4 or KSHV K5 protein induced BST-2 down-regulation. 2-thio-6-azauridine suppresses HIV-1 production in a BST-2-dependent manner. Further results indicate that 2-thio-6-azauridine does not interrupt the interaction of BST-2 with Vpu and ß-TrCP2, but decreases BST-2 ubiquitination. CONCLUSION: Our study demonstrates the feasibility of using small molecules to target Vpu function and sensitize wild type HIV-1 to BST-2-mediated host restriction.

Inhibition of human coronavirus NL63 infection at early stages of the replication cycle.

Human coronavirus NL63 (HCoV-NL63), a recently discovered member of the Coronaviridae family, has spread worldwide and is associated with acute respiratory illness in young children and elderly and immunocompromised persons. Further analysis of HCoV-NL63 pathogenicity seems warranted, in particular because the virus uses the same cellular receptor as severe acute respiratory syndrome-associated coronavirus. As there is currently no HCoV-NL63-specific and effective vaccine or drug therapy available, we evaluated several existing antiviral drugs and new synthetic compounds as inhibitors of HCoV-NL63, targeting multiple stages of the replication cycle. Of the 28 compounds that we tested, 6 potently inhibited HCoV-NL63 at early steps of the replication cycle. Intravenous immunoglobulins, heptad repeat 2 peptide, small interfering RNA1 (siRNA1), siRNA2, beta-D-N(4)-hydroxycytidine, and 6-azauridine showed 50% inhibitory concentrations of 125 microg/ml, 2 microM, 5 nM, 3 nM, 400 nM, and 32 nM, respectively, and low 50% cytotoxicity concentrations (>10 mg/ml, >40 microM, >200 nM, >200 nM, >100 microM, and 80 microM, respectively). These agents may be investigated further for the treatment of coronavirus infections.

[Biotechnological synthesis of ribavirin. Effect of ribavirin and its various combinations on the reproduction of Vaccinia virus]. / Biotekhnologicheskii sposob polucheniia ribavirina. Deistvie ribavirina i nekotorykh ego kombinatsii na reproduktsiiu virusa ospovaktsiny (Vaccinia virus).

The biotechnological method of synthesis of ribavirin, vidarabin, and 6-azauridine by the use of immobilized recombinant enzymatic preparations of nucleoside phosphorylase was improved. The effect of ribavirin and its combinations with the other synthesized nucleosides on the reproduction of Vaccinia virus was studied using cultures of Vero cells. The combination of ribavirin and vidarabin was shown to provide an antiviral effect at lesser concentrations than when these compounds were taken separately. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

A simple assay for determining antiviral activity against Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that is emerging as a significant human pathogen in many regions of the world, including Africa, Asia, and Europe. In this report, we describe a simple screening method for discovering new antiviral compounds directed against CCHFV. Antiviral activity was determined by assaying infected SW-13 cells (human adrenal gland carcinoma) for protection from cytopathic effect (CPE). By using an in vitro neutral red uptake assay, we were able to quantitatively measure CPE induced by CCHFV. As a proof of concept, we used this method to evaluate the antiviral activity of ribavirin and a series of structural analogs (ribamidine, 6-azauridine, selenazofurin, and tiazofurin) against four geographically diverse strains of CCHFV. Ribavirin inhibited the replication of CCHFV as reported previously using plaque reduction assays. One drug, ribamidine, showed antiviral activity that was 4.5- to 8-fold less than that of ribavirin, and the other three drugs (6-azauridine, selenazofurin, and tiazofurin) did not show significant antiviral activity. There were no significant differences in drug sensitivities among the CCHFV strains. Development of this simple and reliable assay will potentially allow high-throughput screening for discovering additional antiviral drugs to combat this important public health threat.

Interferon, ribavirin, 6-azauridine and glycyrrhizin: antiviral compounds active against pathogenic flaviviruses.

Ribavirin, interferon-alpha (IFN-alpha), 6-azauridine and glycyrrhizin were tested in vitro for their antiviral activities against 11 pathogenic flaviviruses belonging to principal antigenic complexes or individual serogroups of medical importance: dengue, Japanese encephalitis, mammalian tick-borne and yellow fever virus (YFV) groups. Antiviral activity was estimated by the reduction of the cytopathic effect of each flavivirus in Vero cells and by the reduction in virus titer. Cytotoxicity was evaluated by determining the inhibition of Trypan blue exclusion in confluent cell cultures and by the evaluation of the inhibitory effect on cell growth. The specificity of action of each tested compound was estimated by the selectivity index (CC(50)/EC(50)). IFN-alpha proved to be a selective and potent inhibitor of the replication of the 11 tested pathogenic flaviviruses. Ribavirin and 6-azauridine proved to be active on the replication of the 11 tested pathogenic flaviviruses at the concentrations which did not alter normal cell morphology, but they were not selective inhibitors when selectivity indices were evaluated with regard to the inhibition of cell growth because of their cytostatic effect. Glycyrrhizin inhibited the replication of flaviviruses at high non-cytotoxic concentrations. These antiflavivirus compounds should be further evaluated for their efficacy in the treatment of flavivirus infections in vivo.

Identification of active antiviral compounds against a New York isolate of West Nile virus.

The recent West Nile virus (WNV) outbreak in the United States has increased the need to identify effective therapies for this disease. A chemotherapeutic approach may be a reasonable strategy because the virus infection is typically not chronic and antiviral drugs have been identified to be effective in vitro against other flaviviruses. A panel of 34 substances was tested against infection of a recent New York isolate of WNV in Vero cells and active compounds were also evaluated in MA-104 cells. Some of these compounds were also evaluated in Vero cells against the 1937 Uganda isolate of the WNV. Six compounds were identified to be effective against virus-induced CPE with 50% effective concentrations (EC50) less than 10 microg/ml and with a selectivity index (SI) of greater than 10. Known inhibitors of orotidine monophosphate decarboxylase and inosine monophosphate dehydrogenase involved in the synthesis of GTP, UTP, and TTP were most effective. The compounds 6-azauridine, 6-azauridine triacetate, cyclopententylcytosine (CPE-C), mycophenolic acid and pyrazofurin appeared to have the greatest activities against the New York isolate, followed by 2-thio-6-azauridine. Anti-WNV activity of 6-azauridine was confirmed by virus yield reduction assay when the assay was performed 2 days after initial infection in Vero cells. The neutral red assay mean EC50 of ribavirin was only 106 microg/ml with a mean SI of 9.4 against the New York isolate and only slightly more effective against the Uganda isolate. There were some differences in the drug sensitivities of the New York and Uganda isolates, but when comparisons were made by categorizing drugs according to their modes of action, similarities of activities between the two isolates were identified.

Characteristics of the anticoccidial activity of 6-azauracil.

The characteristics of anticoccidial activities of 6-azauracil (AzU) were investigated in the battery trials utilizing the White Leghorn cockerels, which were infected with Eimeria tenella, E. necatrix, E. acervulina, E. maxima and/or E. brunetti. AzU was mixed into the basal starter feed and fed ad libitum to the birds from 1 day before the inoculation of oocysts to the time of necropsy. AzU showed remarkable anticoccidial activity against E. tenella and E. necatrix infections at doses of 1,000 ppm in feed or more, and fairly good effect against E. acervulina infection at the dose of 4,000 ppm in feed. Inadequate effect against E. maxima infection and null in effect against E. brunetti at the dose of 4,000 ppm were resulted for this drug. The effect of 6-azauridine, ribonucleoside of AzU against E. tenella and E. necatrix infections at 1,000 ppm was tested and revealed negative results. In the test with AzU-resistant line of E. tenella, cross resistance was confirmed between AzU and emimycin riboside, a uridine analogue. Activity of AzU was studied in vitro against E. tenella in chick kidney cells. AzU inhibited the development of the first- and second-generation schizonts at 100-200 ppm in the medium. Degenerated parasites were observed.

Acquisition of resistance to 6-azauridine through DNA amplification in neoplastic but not normal osteoblasts.

In this communication, we have characterized the resistance to AZUrd in tumorigenic mouse C3H-OS osteosarcoma cells and non-tumorigenic MC3T3-E1 osteoblast cells. DNA and RNA blot analysis showed a 30-fold increase in UMP synthase specific DNA and a 10-fold increase in mRNA, respectively, in resistant versus non-resistant C3H-OS cells. No corresponding increases in either UMP synthetase DNA or mRNA were evident in resistant MC3T3-E1 osteoblasts. Karyotype analysis of MC3T3-E1 and C3H-OS cells revealed translocations in the resistant cells. Regardless of drug-sensitive or resistant phenotype, the normal and neoplastic cells exhibited aneuploidy which was significantly more pronounced in the non-resistant tumor cells. Additionally, the number of chromosomes decreased in all resistant cells whether normal or neoplastic. We conclude that genomic instability in neoplastic cells is a prerequisite for the generation of drug resistant variants via the process of gene amplification.

Gene amplification and chromosome rearrangements: a study of a single cell lineage selected for amplification and deamplification of the UMP synthase gene.

The UMP synthase gene is stably amplified in Chinese hamster lung cells selected for resistance to pyrazofurin (PF) and 6-azauridine (6AUR), inhibitors of the decarboxylase activity of the bifunctional UMP synthase enzyme. The amplified DNA is located intrachromosomally as expanded chromosomal regions (ECRs). Growth of these cells in 5-fluorouracil enables rapid selection of cells that have undergone deamplification and consequently lost resistance to PF + 6AUR. Detailed cytogenetic analyses and fluorescence in situ hybridization on three consecutive amplification-deamplification cycles in descendants of the same cloned cell showed a unique position and structure for the ECR. In the first cycle of amplification, the ECR forms a homogeneously staining region on a small marker chromosome (M3). In the second cycle of amplification, a chromosomal break was noted at the site of the endogenous UMP synthase gene on another derivative chromosome, M2, with amplification resulting in an abnormally banded region on a third marker (M3). The third cycle of amplification produced a cell line with an ECR on the distal portion of M2. This ECR was unstable, showing variations in size as well as translocations and other chromosome rearrangements. Our data, taken in its entirety, suggest a relationship between amplification as an ECR and chromosome rearrangements in Chinese hamster cells.