Journal Article•
Phase Specificity of 5-Azacytidine against Mammalian Cells in Tissue Culture
TL;DR: 5-Azacytidine significantly inhibited mitosis of L1210 cells in culture only after 2 hr of incubation and was correlated to the inhibition of DNA synthesis, indicating that 5-azacyTidine kills cells in certain phases of the cell cycle.
Abstract: Summary 5-Azacytidine (1 to 5 μg/ml) significantly inhibited mitosis of L1210 cells in culture only after 2 hr of incubation The inhibition of mitosis was correlated to the inhibition of DNA synthesis With asynchronous DON and L1210 cells, the cell-kill (inhibition of proliferative capacity) reached a saturation value at high doses of the drug Any further increase in drug concentration did not result in a corresponding increase in percentage of cell-kill This indicated that 5-azacytidine kills cells in certain phases of the cell cycle With synchronous DON cells, it was shown that the drug was lethal predominantly in the S phase 5-Azacytidine caused considerable chromosome damage when L1210 cells in culture were exposed to the drug at 5 μg/ml for 2 hr at 37°
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TL;DR: Azacitidine, the first drug approved by the U.S. FDA for MDS, has a favorable safety profile and provides a clinical benefit of eliminating transfusion dependence and complete or partial normalization of blood counts and bone marrow blast percentages in responding patients.
Abstract: On May 19, 2004, azacitidine (5-azacytidine; Vidaza(trade mark); Pharmion Corporation, Boulder, CO, http://www.pharmion.com) for injectable suspension received regular approval by the U.S. Food and Drug Administration (FDA) for the treatment of all subtypes of myelodysplastic syndrome (MDS). This report summarizes the basis for this approval. Effectiveness was demonstrated in one randomized, controlled trial comparing azacitidine administered s.c. with best supportive care (observation group) and in two single-arm studies, one in which azacitidine was administered s.c. and in the other in which it was administered i.v. The dose of azacitidine, 75 mg/m2/day for 7 days every 28 days, was the same in all three studies. In the randomized trial, study participants were well matched with respect to age, sex, race, performance status, MDS subtype, and use of transfusion during the 3 months before study entry. Patients in the observation arm were permitted by protocol to cross over to azacitidine treatment if their disease progressed according to prespecified criteria. During the course of the study, more than half of the patients in the observation arm did cross over to the azacitidine treatment arm. The primary efficacy end point was the overall response rate. Response consisted of complete or partial normalization of blood cell counts and of bone marrow morphology. The response rate in the azacitidine arm was about 16%; there were no responses in the observation arm. The response rates in the two single-arm studies were similar (13% and 19%). The responses were sustained, with median durations of 11 months and 17 months respectively. Responding patients who were transfusion dependent at study entry lost the need for transfusions. In addition, about 19% of patients had less than partial responses (termed improvement), and two-thirds of them became transfusion independent. Common adverse events associated with azacitidine treatment were gastrointestinal (nausea, vomiting, diarrhea, constipation, and anorexia), hematologic (neutropenia, thrombocytopenia), fevers, rigors, ecchymoses, petechiae, injection site events, arthralgia, headache, and dizziness. Liver function abnormalities occurred in 16% of patients with intercurrent hepatobiliary disorders and in two patients with previously diagnosed liver cirrhosis. Renal failure occurred in patients during sepsis and hypotension. There were no deaths attributed to azacitidine. Azacitidine, the first drug approved by the U.S. FDA for MDS, has a favorable safety profile and provides a clinical benefit of eliminating transfusion dependence and complete or partial normalization of blood counts and bone marrow blast percentages in responding patients.
419 citations
Cites background from "Phase Specificity of 5-Azacytidine ..."
...Azacitidine is most toxic during the S-phase of the cell cycle, but the predominant mechanism of cytotoxicity has not been established [ 3 , 4]....
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Journal Article•
TL;DR: This study attempts to determine the primary actions of 5-azacytidine (5-azaCR) on L1210 leukemia and finds that it inhibits the incorporation of tritiated thymidine or deoxyadenosine into DNA to a greater extent than it inhibitsThe incorporation oftritiated uridine into RNA.
Abstract: This study attempts to determine the primary actions of 5-azacytidine (5-azaCR) on L1210 leukemia. This agent was cytotoxic toward L1210 cells growing in culture with 50 and 90% inhibition dose values of 0.019 and circa 0.15 μg/ml, respectively. 5-AzaCR inhibits the incorporation of tritiated thymidine or deoxyadenosine into DNA to a greater extent than it inhibits the incorporation of tritiated uridine into RNA. Similar results were obtained with ascitic cells isolated from leukemic mice. Equimolar amounts of cytidine reduced the inhibition of DNA synthesis, as well as the inhibition of cell growth in culture caused by 5-azaCR. Uridine, but not deoxycytidine or deoxyuridine, was also effective, but to a lesser extent than was cytidine. With cell-free extracts isolated from L1210 cells in culture, no significant effect was found on enzyme systems directly or indirectly involved in DNA synthesis.
With 5-azaCR-4-14C as the precursor, this agent was found to be phosphorylated in all leukemic tissues studied. The majority of phosphorylated products existed as the triphosphate in ascitic and cultured L1210 cells. A portion (10 to 20%) of all these phosphorylated derivatives appeared to be further reduced to deoxyribonucleoside di- and/or triphosphate forms. 5-AzaCR was also incorporated into polynucleotides in all tissues studied and incorporated into both RNA (80 to 90% total incorporated radioactivity) and DNA fractions (10 to 20%) in L1210 cells in culture. In the presence of cytidine, phosphorylation of 5-azaCR, subsequent reduction, and incorporation into polynucleotides were greatly inhibited.
A probable mechanism of action of 5-azaCR on L1210 leukemia is proposed.
266 citations
Cites result from "Phase Specificity of 5-Azacytidine ..."
...However, by comparing the inhibition of DNA synthesis and that of mitosis of LI210 cells in culture for the same period of incubation, the results are rather compatible (19)....
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TL;DR: It is strongly suggested that S-phase-specific cytotoxic/cytostatic drugs increase fetal hemoglobin by a mechanism that does not involve inhibition of DNA methylation.
Abstract: The increase in fetal-hemoglobin synthesis in patients with beta-thalassemia or sickle-cell anemia induced by 5-azacytidine has been attributed to hypomethylation of DNA in the region of the gamma-globin genes. To determine whether hydroxyurea, a cytotoxic/cytostatic drug that does not influence DNA methylation, might stimulate fetal-hemoglobin synthesis, we phlebotomized two juvenile cynomolgus monkeys to induce anemia and reticulocytosis and then treated them with hydroxyurea. Immediately after phlebotomy was initiated, there was a rise in the level of F cells, which stabilized at an average value of 13 per cent in one animal and 20 per cent in the other during a two-month control period. Fetal hemoglobin gradually rose from undetectable values before bleeding to 3 per cent in one animal and 5 per cent in the other. Sixty-two days after initiation of phlebotomy, hydroxyurea (50 mg per kilogram of body weight per day for five days) induced only a small and transient increase in F cells and fetal hemoglobin. Two weeks later, however, a similar course (100 mg per kilogram per day) resulted in a prompt and dramatic increase in both indexes. These results strongly suggest that S-phase-specific cytotoxic/cytostatic drugs increase fetal hemoglobin by a mechanism that does not involve inhibition of DNA methylation.
221 citations
TL;DR: Clinical results in solid tumors are not encouraging, but 5-azacytidine shows consistent antitumor activity in patients with acute myelogenous leukemia resistant to previous treatment.
Abstract: Clinical studies involving 5-azacytidine, a ring analogue of cytidine, began in Europe in 1967 and the United States in 1970, and we review available preclinical and clinical studies here. The drug possesses cytotoxic, antimicrobial, antineoplastic, abortive, and mutagenic activity in various biological systems. 5-Azacytidine is thought to exert its antineoplastic effect through interference with nucleic acid metabolism. The dose-limiting toxicities are nausea, vomiting, and leukopenia, while the incidence of thrombocytopenia is low. Hepatic toxicity ranges from abnormal findings in liver function tests to hepatic coma. Clinical results in solid tumors are not encouraging, but 5-azacytidine shows consistent antitumor activity in patients with acute myelogenous leukemia resistant to previous treatment. An overall response rate of 36%, with 20% complete remissions, was achieved in 200 previously treated patients with acute myelogenous leukemia. Further studies must define the role of 5-azacytidine alone and in combination for the first-line treatment of acute myelogenous leukemia.
183 citations
TL;DR: While at least part of decitabine's activity is through induction of hypomethylation and reactivation of critical genes, mechanisms independent from hypometHylation are also important for decitABine's antitumor activity.
Abstract: Purpose of the review Epigenetic changes marked by DNA methylation are known to contribute to the malignant transformation of cells by silencing critical genes. Decitabine inhibits DNA methyltransferase and has shown therapeutic effects in patients with hematologic malignancies. However, the connection between the clinical activity of decitabine and its demethylating activity is not clear. Herein, we summarize the results of recent clinical trials of decitabine in hematologic malignancies, and review the translational research into decitabine's mechanism of clinical activity. Recent findings Low-dose decitabine has been studied recently in multiple clinical trials and has been shown to be effective for treatment of myelodysplastic syndromes. Correlative laboratory studies of clinical trials have shown that decitabine induces global hypomethylation as well as hypomethylation of gene-specific promoters and activation of gene expression. Past a given threshold, induction of higher degrees of hypomethylation is not directly associated with a better clinical outcome. Moreover, studies have suggested that patients with promoter hypermethylation of p15 INK4B at baseline have paradoxically a lower chance of achieving response than those without hypermethylation. Furthermore, several other genes activated by decitabine were independent of hypomethylation in the promoter regions. Conclusion While at least part of decitabine's activity is through induction of hypomethylation and reactivation of critical genes, mechanisms independent from hypomethylation are also important for decitabine's antitumor activity.
167 citations
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"Phase Specificity of 5-Azacytidine ..." refers methods in this paper
...Such data were obtained with presumably phase-specific agents such as Cytosar by Karon and Shirakawa (9) and with vinblastine, TdR-3H, and hydroxyurea by Bruce et al. (2, 3)....
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...Mean transit times through the cell cycle of both cell lines were determined from plots of labeled mitoses at various times after pulse labeling with TdR-3H (15). •This investigation was supported in part by Contract PH43-68-1023 with Chemotherapy, National Cancer Institute, NIH, Bethesda, Md. 2The abbreviations used are: 5-azaCR, 5-azacytidine; TdR, thy niidine; MI, mitotic index....
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...The incorporation of radioactive precursors, e.g., TdR-3H and uridine-3H, into respective macromolecules of intact LI210 cells was determined by the method described elsewhere (10)....
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...Cells (circa 1 X IO6 cells/8-oz. prescription bottle) were incubated with 5-azaCR and labeled metabolite (TdR-3H or UR-3H, 6.4 MCi/4.6 ng/ml of reaction system) at 37°for 2 to 8 hr....
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...When the synchronous culture was pulse-labeled with TdR-3H the percentage of labeled cells was 0, 72, 85, 88, 90, 62, and 44% at 1, 2, 3, 4, 6, 8, and 10 hr, respectively, after planting mitotic cells....
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Journal Article•
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...(2, 3) have shown that with phase-specific...
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Journal Article•
TL;DR: Although the length of the cell cycle varies in different kinds of cells, it is shorter in certain cells of the adult animal than in some of the fastest growing tumors, so tumor growth must involve other kinetic parameters besides speed of cell proliferation.
Abstract: Summary Metabolic activities of cells during interphase can be studied by high-resolution autoradiography with tritium-labeled precursors of nucleic acids and proteins. After completion of mitosis, offspring of the parent cell may divide again, or may differentiate and die without further division. Dividing cells go through a series of metabolic changes which constitute the cell cycle. The cell cycle is subdivided into 4 distinct phases: ( a ) a postmitotic phase called G 1 , during which the cell synthesizes ribonucleic acid (RNA) and proteins; ( b ) an S phase, during which the amount of deoxyribonuleic acid (DNA) is duplicated while protein and RNA syntheses continue; ( c ) a postsynthetic phase, called G 2 , characterized by no net synthesis of DNA and continued RNA and protein synthesis as in postmitotic phase; and ( d ) mitosis, extending from beginning of prophase to completion of telophase during which there is no DNA synthesis, protein synthesis is reduced to a minimum, and RNA synthesis is limited to early prophase and late telophase. The length of the cell cycle and its 4 phases can be obtained by appropriate experimentation with labeled thymidine, a precursor of DNA, and high-resolution autoradiography. Although the length of the cell cycle varies in different kinds of cells, it is shorter in certain cells of the adult animal than in some of the fastest growing tumors. Tumor growth, therefore, must involve other kinetic parameters besides speed of cell proliferation. Since these parameters are related to control of cell division and since a cell in DNA synthesis is generally a cell that has already taken the decision to divide, the various factors involved in initiation of DNA biosynthesis are of particular interest in any attempt to explain the mechanism of tumor growth. It is possible that the initiation of DNA biosynthesis may involve DNA itself, and may be mediated through synthesis of specific RNA and enzymes.
267 citations
"Phase Specificity of 5-Azacytidine ..." refers background in this paper
...Some antitumor agents have selective biological and/or biochemical effects (1, 8, 9, 11-13, 16, 21) at certain phases of the cell cycle....
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