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.

Oligonucleotides as modulators of cancer gene expression.

The delineation of gene function has always been an intensive subject of investigations. Recent advances in the synthesis and chemistry of oligonucleotides have now made these molecules important tools to study and identify gene function and regulation. Modulation of gene expression using oligonucleotides has been targeted at different levels of the cellular machinery. Triplex forming oligonucleotides, as well as peptide nucleic acids, have been used to inhibit gene expression at the level of transcription; after binding of these specific oligonucleotides, conformational change of the DNA's helical structure prevents any further DNA/protein interactions necessary for efficient transcription. Gene regulation can also be achieved by targeting the translation of mRNAs. Antisense oligonucleotides have been used to down-regulate mRNA expression by annealing to specific and determined region of an mRNA, thus inhibiting its translation by the cellular machinery. The exact mechanism of this type of inhibition is still under intense investigation and is thought to be related to the activation of RNase H, a ribonuclease that is widely available that can cleave the RNA/DNA duplex, thus making it inactive. Another well-characterized means of interfering with the translation of mRNAs is the use of ribozymes. Ribozymes are small catalytic RNAs that possess both site specificity and cleavage capability for an mRNA substrate, inhibiting any further protein formation. This review describes how these different oligonucleotides can be used to define gene function and discusses in detail their chemical structure, mechanism of action, advantages and disadvantages, and their applications.

Comparison of the induction of apoptosis in human leukemic cell lines by 2',2'-difluorodeoxycytidine (gemcitabine) and cytosine arabinoside.

The induction of DNA fragmentation by cytosine arabinoside (araC) and 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) was compared in human leukemic cell lines. For both araC and dFdC this process was time- and concentration-dependent and resulted in loss of clonogenic survival of HL-60 myeloid leukemic cells. There was a marked difference in the potency between these two analogs in inducing apoptosis. A 6 h exposure to 5 microM araC was required to produce DNA laddering in HL-60 cells, whereas dFdC at a concentration 100-fold less (0.05 microM) was sufficient to produce similar results. Pre-incubation of HL-60 cells with staurosporine, a non-specific protein kinase C inhibitor, increased the level of apoptosis induced by a 3 h exposure to araC or dFdC, suggesting the possible involvement of this family of enzymes in this process. Also, dFdC was able to increase the expression of both c-jun and c-fos in Molt-3 leukemic cells with a concentration known to induce apoptosis in this cell line.

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.

Kinetic studies on 2',2'-difluorodeoxycytidine (Gemcitabine) with purified human deoxycytidine kinase and cytidine deaminase.

Phosphorylation of cytosine analogs by deoxycytidine kinase (dCK) and deamination by cytidine deaminase (CDA) are two important processes in the activation and elimination of these drugs. We have investigated the kinetic parameters of 2',2'-difluorodeoxycytidine (dFdC) using purified enzymes from human cells. Deoxycytidine (CdR) and dFdC had Km values of 1.5 and 4.6 microM for dCK, respectively. Feedback inhibition of dCK by deoxycytidine 5'-triphosphate (dCTP) was also studied. Our results show that dCTP produced a greater inhibition of the phosphorylation of dFdC than CdR with concentrations of dCTP ranging from 1 to 25 microM. dFdC was a good substrate for CDA. Kinetic studies with this enzyme gave Km values for CdR and dFdC of 46.3 and 95.7 microM, respectively. The effect of competitive inhibitors of CDA on the deamination of dFdC was also investigated. Diazepinone riboside was a more potent inhibitor than tetrahydrouridine using either CdR or dFdC as the substrate. Inhibitors of CDA could be useful in clinical trials in patients with cancer to increase the chemotherapeutic effectiveness of dFdC.

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.

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.