Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells.

Naturally occurring drug resistance genes of human origin can be exploited for selection of genetically engineered cells co-expressing a desired therapeutic transgene. Their non-immunogenicity in clinical applications would be a major asset. Human cytidine deaminase (hCD) is a chemoresistance gene that inactivates cytotoxic cytosine nucleoside analogs, such as cytosine arabinoside (Ara-C). The aim of this study was to establish if the hCD gene can serve as an ex vivo dominant selectable marker in engineered bone marrow stromal cells (MSCs). A bicistronic retrovector comprising the hCD cDNA and the green fluorescent protein (GFP) reporter gene was generated and used for transduction of A549 cells and primary murine MSCs. Analysis of transduced cells demonstrated stable integration of proviral DNA, more than 1000-fold increase in CD enzyme activity, and drug resistance to cytosine nucleoside analogs. In a mixture of transduced and untransduced MSCs, the percentage of retrovector-expressing cells could be increased to virtual purity (>99.5%) through in vitro drug selection with 1 microM Ara-C. Increased selective pressure with 2.5 microM Ara-C allowed for enrichment of a mixed population of MSCs expressing approximately six-fold higher levels of GFP and of CD activity when compared with unmanipulated engineered MSCs. Moreover, engraftment and endothelial differentiation of these in vitro selected and enriched gene-modified marrow stromal cells was demonstrated by Matrigel assay in vivo. In conclusion, these findings outline the potential of human CD as an ex vivo selection and enrichment marker of genetically engineered MSCs for transgenic cell therapy applications.

DNA methylation of retinoic acid receptor beta in breast cancer and possible therapeutic role of 5-aza-2'-deoxycytidine.

The retinoic acid receptor beta (RARbeta), a putative tumor suppressor gene, has been reported to be poorly expressed in breast cancer. In this report using the methylation-specific PCR reaction we observed DNA methylation in the promoter region of RARbeta in several primary breast tumors. DNA sequence analysis showed that the positions of 5-methylcytosine in the RARbeta promoter region was almost identical to that reported previously by our laboratory for human DLD-1 colon carcinoma cells (Anti-Cancer Drugs 1998; 9: 743). Several other cancer-related genes have been also reported to be silenced by DNA methylation, including the p16 tumor suppressor gene, E-cadherin, an invasion suppressor gene and the estrogen receptor gene in breast cancer cell lines. Since breast cancer cells have several potential target genes for the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-Aza-CdR), we investigated the in vitro antineoplastic activity of this analog on the human breast cancer cell line MDA-MB-231. We report that 5-Aza-CdR is a potent growth inhibitor and a potent cytotoxic agent against the breast carcinoma cells. These results suggest that 5-Aza-CdR may be an interesting agent to investigate in patients with breast cancer resistant to conventional chemotherapy.

Retroviral transfer and long-term expression of human cytidine deaminase cDNA in hematopoietic cells following transplantation in mice.

The chemotherapeutic effectiveness of cytosine nucleoside analogues used in cancer therapy is limited by their dose-dependent myelosuppression. A way to overcome this problem would be to insert the drug-resistance gene, cytidine deaminase (CD), into normal hematopoietic cells. CD catalyzes the deamination and pharmacological inactivation of cytosine nucleoside analogues, such as cytosine arabinoside (Ara-C). The objective of this study was to determine if we could obtain long-term persistence and expression of proviral CD in hematopoietic cells following transplantation of CD-transduced bone marrow cells in mice. Murine hematopoietic cells were transduced with an MFG retroviral vector containing CD cDNA and transplanted into lethally irradiated mice. The recipient mice were administered three courses of 10-15 h i.v. infusions of Ara-C (75-110 mg/kg). Blood, marrow and spleen samples were obtained and analyzed for CD proviral DNA by PCR, CD activity by enzyme assay, and drug resistance to Ara-C by clonogenic assay. We detected the presence of the CD proviral DNA in most of the samples examined. Approximately 1 year after transplantation several mice showed increased expression of CD activity in these tissues and some mice displayed signs of Ara-C resistance. These data demonstrate that persistent in vivo expression of proviral CD can be achieved in transduced hematopoietic cells and indicate some potential of this gene for chemoprotection to improve the efficacy of cytosine nucleoside analogues in cancer therapy.

Pilot phase I-II study on 5-aza-2'-deoxycytidine (Decitabine) in patients with metastatic lung cancer.

5-Aza-2'-deoxycytidine (5-AZA-CdR, Decitabine) is a nucleoside analog and an active drug for the therapy of acute leukemia. The incorporation of 5-AZA-CdR into DNA blocks DNA methylation and can result in the activation of specific genes, such as tumor suppressor genes. This novel mechanism of action of 5-AZA-CdR stimulated our interest in its potential for cancer therapy in patients with lung cancer. Using a colony assay we observed that 5-AZA-CdR showed a potent antineoplastic effect against two human lung carcinoma cell lines. The objective of this preliminary phase I-II study was to evaluate the toxicity and clinical efficacy of 5-AZA-CdR in patients with stage IV non-small cell lung carcinoma. There were 15 patients that entered the clinical study. For nine assessable patients that received 5-AZA-CdR by a single 8 h i.v. infusion of 200-660 mg/m2 for one or more cycles, the median survival duration was 6.7 months, with three patients surviving more than 15 months. The steady-state plasma concentration of 5-AZA-CdR during the infusion was estimated in some patients and was in the same range that produced activation of a tumor suppressor gene in human lung tumor cell lines as reported by other investigators. The major side effect of 5-AZA-CdR was hematopoietic toxicity which required a 5-6 week recovery period before the next cycle of therapy. This study suggests that 5-AZA-CdR may have some clinical activity against metastatic lung carcinoma using this type of dose schedule.

Enhancement of the antileukemic activity of 5-aza-2'-deoxycytidine by cyclopentenyl cytosine in HL-60 leukemic cells.

We have investigated the capacity of cyclopentenyl cytosine (CPE-C), a potent inhibitor of CTP synthetase, to modulate the antineoplastic activity of 5-aza-2'-deoxycytidine (DAC) on HL-60 myeloid leukemic cells. The combination of CPE-C and DAC produced an additive effect on the growth inhibition of the cells following a treatment of 48-96 h. Cytotoxicity experiments measured by the cloning of cells in soft agar following 24 and 48 h exposures produced a more than additive effect when the drugs were used in combination. Evaluation of the effect of CPE-C and DAC on the induction of differentiation of HL-60 cells following a 48 h treatment revealed that the combination of the drugs produced a more than additive effect than when the drugs were used alone. Measurement of the intracellular pool of deoxycytidine triphosphate (dCTP) showed that a 6 h exposure to 0.05 and 0.1 microM of CPE-C reduced the pool by 60 and 88%, respectively. The decrease in the dCTP pool was correlated with a higher incorporation of radioactive DAC into DNA. The deamination of CPE-C to cyclopentenyl uridine by cytidine deaminase was investigated with the purified enzyme from human placenta. We report here that CPE-C is a very poor substrate for cytidine deaminase as compared with cytidine. These studies suggest that CPE-C could be used as a biochemical modulator to increase the antileukemic action of DAC.

Interaction of 5-aza-2'-deoxycytidine with amsacrine or 1,25-dihydroxyvitamin D3 on HL-60 myeloid leukemic cells and inhibitors of cytidine deaminase.

5-aza-2'-deoxycytidine (5-Aza-CdR) is an experimental anti-leukemic agent that can induce the differentiation of myeloid leukemic cells and is currently under investigation in clinical trials in patients with leukemia. The objective of this study was to investigate the in vitro interaction of 5-Aza-CdR with different agents which may increase its clinical efficacy. Since 5-Aza-CdR has been used in combination with amsacrine in patients with relapsed leukemia, we have investigated the in vitro antileukemic activity of these agents in combination on human HL-60 myeloid leukemic cells. For a 24-hour drug exposure, we observed that the interaction of 5-Aza-CdR with amsacrine was subadditive to was synergistic with respect to the loss of clonogenicity of the HL-60 leukemic cells as demonstrated by a colony assay. We have also investigated the interaction of 5-Aza-CdR with 1,25-dihydroxyvitamin D3, an agent that can induce in vitro differentiation of myeloid leukemic cells. The combination of 5-Aza-CdR with 1,25-dihydroxyvitamin D3 produced a synergistic reduction in the clonogenicity of the HL-60 leukemic cells. These results indicate that 5-Aza-CdR can interact synergistically with both cytotoxic agents and agents that induce differentiation, and can provide some rationale for clinical investigations on these combinations in patients with acute leukemia. We have also explored other approaches to increase the clinical effectiveness of 5-Aza-CdR by investigating the in vitro effect of different inhibitors of human cytidine deaminase, the enzyme responsible for the rapid in vivo inactivation of this analogue.

Enhancement of anti-neoplastic activity of cytosine arabinoside against human HL-60 myeloid leukemic cells by 3-deazauridine.

Drug resistance is one of the major reasons for failure of chemotherapy of acute leukemia with cytosine arabinoside (ARA-C). In order to overcome this problem we have investigated the interaction of ARA-C with 3-deazauridine (3-DU) against HL-60 myeloid leukemic cells. 3-DU is an interesting agent to use in combination with ARA-C, since drug-resistant cells that are deficient in deoxycytidine kinase are very sensitive to this uridine analogue. We have observed that for both short and long drug exposure there was a potent synergistic interaction between ARA-C and 3-DU with respect to their cytotoxic effects on HL-60 leukemic cells. This synergy could be explained by an increased cellular uptake of ARA-C to ARA-CTP by the leukemic cells in the presence of 3-DU, due to the reduction in the pool of dCTP produced by this latter analogue. Since dCTP is a potent feedback inhibitor of the phosphorylation of ARA-C by deoxycytidine kinase, the reduction in the dCTP produced by 3-DU results in an increased rate of phosphorylation of the arabinosyl analogue. Our results suggest that ARA-C and 3-DU may be an interesting drug combination to circumvent drug resistance in the chemotherapy of acute leukemia.

Comparison of antineoplastic activity of 2',2'-difluorodeoxycytidine and cytosine arabinoside against human myeloid and lymphoid leukemic cells.

2',2'-difluorodeoxycytidine (known as dFdC, Gemcitabine and LY188011) is a new analog of deoxycytidine which has demonstrated excellent antineoplastic activity against many kinds of solid tumors and leukemic cell lines. We were interested in the comparison of the antineoplastic activity of this new antimetabolite with cytosine arabinoside (ARA-C) against HL-60 myeloid, RPMI-8392 B-lymphoid and Molt-3 T-lymphoid leukemic cell lines. Our in vitro experiments showed that dFdC was a more potent cytostatic drug than ARA-C against all the leukemic lines with IC50 ranging from 3 to 10 nM for dFdC and from 26 to 52 nM for ARA-C for a 48 h exposure. The cytotoxicity of both drugs was evaluated by clonogenic assay and dFdC was found to be 100 times more potent than ARA-C against all the leukemic cell lines for both a 2 h and a 24 h exposure. The recovery of DNA synthesis after drug removal was much slower for dFdC than for ARA-C. However, in contrast to cytostatic and cytotoxicity results ARA-C was a more potent inhibitor of DNA synthesis than dFdC for all the leukemic cell lines for short exposure. Uptake and elimination of the drugs showed that dFdC accumulated to a higher degree in the leukemic cells than ARA-C and that elimination of this difluoro analog was slower than that of ARA-C. These results indicate that dFdC has more potent in vitro antileukemic activity than ARA-C.

Effect of 5-aza-2'-deoxycytidine and retinoic acid on differentiation and c-myc expression in HL-60 myeloid leukemic cells.

The antineoplastic effects of 5-aza-2'-deoxycytidine (5-AZA-CdR) and retinoic acid on human HL-60 myeloid leukemic cells were investigated. 5-AZA-CdR or retinoic acid reduced the clonogenicity and induced the differentiation of HL-60 leukemic cells. The effects produced by 5-AZA-CdR and retinoic acid in combination on clonogenicity and differentiation of HL-60 leukemic cells were additive. These agents in combination also produced an additive decrease in the mRNA expression of the c-myc. These data indicate that 5-AZA-CdR and retinoic acid in combination produce an additive antineoplastic effect against HL-60 human myeloid leukemic cells.

Cellular pharmacology of 1-beta-D-arabinofuranosylcytosine in human myeloid, B-lymphoid and T-lymphoid leukemic cells.

The in vitro inhibitory action and metabolism of 1-beta-D-arabinofuranosylcytosine (ara-C) on human myeloid (HL-60), B-lymphoid (RPMI-8392), and T-lymphoid (Molt-3) leukemic cells was compared. Ara-C produced greater inhibitory effects in Molt-3 cells than in either HL-60 or RPMI-8392 cells. At a 48 h exposure, ara-C was 7 and 10 times more cytotoxic to Molt-3 cells than to HL-60 and RPMI-8392 cells, respectively. The total ara-C uptake to nucleotides and the formation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) was about 5 times greater in Molt-3 cells than in either HL-60 or RPMI-8392 cells. The incorporation of ara-C into DNA was also higher in Molt-3 cells than in either HL-60 or RPMI-8392 cells. The mean intracellular half-life of ara-CTP was 31.7, 59.4, and 155 min for RPMI-8392, HL-60, and Molt-3 leukemic cells, respectively. The Km and Vmax values of ara-C for deoxycytidine kinase and the feedback inhibition of this enzyme by ara-CTP in the different leukemic cell lines could not explain the differences in metabolism of this analogue in these cells. These data indicate the increased sensitivity of T-lymphoid leukemic cells to ara-C than as compared with B-lymphoid and myeloid leukemic cells was due to an increased rate of formation and a longer half-life of ara-CTP in the T-cells.

Comparison of antineoplastic activity of cytosine arabinoside and 5-aza-2'-deoxycytidine against human leukemic cells of different phenotype.

A comparison of the cellular and molecular pharmacology of the deoxycytidine analogues cytosine arabinoside (Ara-C) and 5-aza-2'-deoxycytidine (5-AZA-CdR) on human myeloid (HL-60), T-cell (Molt-3) and B-cell (RPMI-8392) leukemic cell lines was investigated. Ara-C was a more potent inhibitor of growth than 5-AZA-CdR. In a colony assay, 5-AZA-CdR was a more potent cytotoxic agent than Ara-C for both the myeloid and B-cell leukemic cells, but not for the T-cell leukemic cells. The total cellular uptake of 5-AZA-CdR was greater than Ara-C for the myeloid and B-cell leukemic cells, whereas for the T-cells the uptake of the arabinosyl analogue was greater. Ara-C produced a potent inhibition of DNA synthesis, whereas no inhibition was detected with 5-AZA-CdR during a short incubation. In contrast, 5-AZA-CdR produced a potent inhibition of DNA methylation whereas Ara-C produced a slight increase in the methylation of DNA. This study shows that there are significant differences in the antineoplastic activity of Ara-C and 5-AZA-CdR against human leukemic cell lines of different phenotype and that these differences are related to differences in the metabolism of these two deoxycytidine analogues and to their effects on DNA synthesis and methylation.

Preclinical evaluation of hematopoietic toxicity of antileukemic agent, 5-aza-2'-deoxycytidine.

The hematopoietic toxicity of 5-aza-2'-deoxycytidine (5-AZA-CdR), an experimental antileukemic agent, was investigated in mice and dogs. Mice were administered a 12 h intravenous infusion of 5-AZA-CdR at a total dose of 20 mg/kg and at different times after treatment samples of bone marrow were analyzed for cell count, DNA synthesis and colony-forming activity (CFUc). The 5-AZA-CdR treatment produced a marked decrease in femur cell count and CFUc activity with recovery occurring on days 8-14. DNA synthesis activity increased significantly during the recovery period. Dogs were also administered a 12 h intravenous infusion at doses 3-7 mg/kg and the blood hemogram evaluated. This treatment produced a pronounced leukopenia, granulocytopenia and thrombocytopenia with a nadir between days 7-16 with recovery occurring on days 19-21. These results indicate that 5-AZA-CdR produces marked hematopoietic toxicity in mice and dogs and this toxicity appears to be reversible under these experimental conditions.

Chemotherapy of L1210 and L1210/ARA-C leukemia with 5-aza-2'-deoxycytidine and 3-deazauridine.

The in vitro and in vivo antineoplastic activity of 5-aza-2'-deoxycytidine (5-AZA-dCyd) and 3-deazauridine (3-DU) against L1210 and L1210/ARA-C (resistant to cytosine arabinoside) leukemic cells were investigated. L1210/ARA-C cells were more sensitive to the inhibitory effects of 3-DU than L1210 cells. Deoxycytidine completely reversed the in vitro cytotoxic effects produced by 3-DU on L1210 cells, but not those produced in L1210/ARA-C cells. L1210/ARA-C cells, which are deficient in deoxycytidine kinase, were completely resistant to the antileukemic effects of 5-AZA-dCyd, whereas this analogue produced a very potent antileukemic effect against L1210 cells. To study the in vivo interaction of 5-AZA-dCyd and 3-DU with respect to drug resistance, mice were simultaneously injected i.v. with 10(4) L1210 cells plus 10(2) L1210/ARA-C cells. A 9-h i.v. infusion of 5-AZA-dCyd (12.8 mg/kg) or 3-DU (186 mg/kg) produced an increase in life span of 56% and 26%, respectively. However, the sequential administration of 5-AZA-dCyd followed by 3-DU produced a 265% increase in life span and 7/10 longterm survivor, a very potent antileukemic effect. These results suggest that 3-DU is an excellent agent for use in combination chemotherapy to overcome drug resistance to the deoxycytidine analogue, 5-AZA-dCyd.

Kinetic interaction of 5-AZA-2'-deoxycytidine-5'-monophosphate and its 5'-triphosphate with deoxycytidylate deaminase.

5-AZA-2'-deoxycytidine-5'-monophosphate (5-AZA-dCMP) was tested as a substrate, and 5-aza-2'-deoxycytidine-5'-triphosphate (5-AZA-dCTP) was tested as an allosteric effector of purified spleen dCMP deaminase. Graphic analysis of the velocity of deamination of 5-AZA-dCMP versus its concentration gave a hyperbolic curve in which the estimated apparent Km was 0.1 mM. Since this curve was not sigmoidal and 5-AZA-dCMP at low concentrations stimulated the rate of deamination of the natural substrate, dCMP, it was proposed that the binding of 5-AZA-dCMP to the allosteric enzyme dCMP deaminase induced the R form. At substrate saturation, the rate of deamination of dCMP was 100-fold greater than that of 5-AZA-dCMP. dTTP inhibited the deamination of 5-AZA-dCMP with first-order kinetics. This inhibition was reversed by either 5-AZA-dCTP or dCTP. However, dCTP alone produced only a weak activation of the deamination of 5-AZA-dCMP in comparison to the potent activation when dCMP was the substrate. 5-AZA-dCTP was just as effective as dCTP for the allosteric activation of the deamination of dCMP. These results indicate that dCMP deaminase can play an important role in the metabolism 5-aza-2'-deoxycytidine nucleotides and may possibly modulate some of the pharmacological activity of this antimetabolite.

Comparison of the antileukemic activity of 5-AZA-2'-deoxycytidine, 1-beta-D-arabinofuranosylcytosine and 5-azacytidine against L1210 leukemia.

The antineoplastic activity and effect on DNA synthesis and DNA methylation of 5-aza-2'-deoxycytidine (5-AZA-CdR), 1-beta-D-arabinofuranosylcytosine (ARA-C), and 5-azacytidine (5-AZA-CR) on L1210 leukemic cells were compared. At equimolar concentrations 5-AZA-CdR produced greater growth inhibition and more cytotoxicity against the L1210 cells than either ARA-C or 5-AZA-CR. ARA-C, but not 5-AZA-CdR or 5-AZA-CR, produced a potent inhibition of DNA synthesis in the L1210 cells. 5-AZA-CdR and 5-AZA-CR, but not ARA-C, inhibited DNA methylation with 5-AZA-CdR being a more effective inhibitor than 5-AZA-CR. At maximum tolerated dose, 5-AZA-CdR produced a much greater increase in life span of mice with L1210 leukemia than ARA-C or 5-AZA-CR. These in vitro and in vivo studies indicate that 5-AZA-CdR is a more potent antineoplastic agent against L1210 leukemic cells than either ARA-C or 5-AZA-CR.

Cell cycle effects and cellular pharmacology of 5-aza-2'-deoxycytidine.

The cytotoxic action of 5-aza-2'-deoxycytidine (5-AZA-CdR) in synchronized cells and logarithmic- and plateau-phase cultures of EMT6 murine tumor cells was investigated. 5-AZA-CdR produced a greater cell kill of S phase cells than of cells in G1 phase. Cells in the logarithmic phase of growth were more sensitive to the cytotoxic effects of 5-AZA-CdR than cells in the plateau phase of growth. These results indicate that 5-AZA-CdR produces a preferential kill of cells in the S phase of the cell cycle. 5-AZA-CdR did not block the cell cycle progression of cells into S phase. The survival curve of EMT6 cells exposed to 5-AZA-CdR suggests that the cytotoxic action of this analogue is not self-limiting. The mutagenic activity of 5-AZA-CdR was investigated using induction of 6-thioguanine resistance in Chinese hamster ovary cells. 5-AZA-CdR was not a detectable mutagen in this assay system.