I n Vitro Metabolism of Deoxycoformycin in Human T Lymphoblastoid Cells PHOSPHORYLATION OF DEOXYCOFORMYCIN AND INCORPORATION INTO CELLULAR DNA*

The biochemical and metabolic effects of deoxycofor- mycin, a potent inhibitor of adenosine deaminase, were investigated using two human T lymphoblastoid cell lines. A dose-response analysis demonstrated that the concentration of deoxycoformycin at which there was 50% inhibition of growth was greater than 1 x M in lymphoblastoid cells. Uptake of deoxycoformycin was biphasic and occurred much more slowly than for natural nucleosides, and lower saturation levels were reached. The intracellular concentration of deoxyco- formycin achieved was 0.4 to 0.5 p~ when the extracellular concentration was 1 p ~ . At 10 ~ L M extracellular concentration, the intracellular concentration was 3-4 p ~ . Although deoxycoformycin at very low concen- trations (1 or 10 FM) did not have any detectable effects on the growth of these cells, the nucleoside was found to be metabolized, and was phosphorylated to give the mono-, di-, and triphosphate derivatives. The triphos- phate derivative was incorporated into cellular DNA with little incorporation into cellular RNA. Metabo- lism of deoxycoformycin in several mutant lymphoblastoid cells deficient in adenosine kinase and/or de- oxycytidine kinase was found to be unchanged from wild-type cells, indicating that these major nucleoside kinases do not play a significant role in the phosphorylation of deoxycoformycin. These results may ac- count,

The biochemical and metabolic effects of deoxycoformycin, a potent inhibitor of adenosine deaminase, were investigated using two human T lymphoblastoid cell lines. A dose-response analysis demonstrated that the concentration of deoxycoformycin at which there was 50% inhibition of growth was greater than 1 x M in lymphoblastoid cells. Uptake of deoxycoformycin was biphasic and occurred much more slowly than for natural nucleosides, and lower saturation levels were reached. The intracellular concentration of deoxycoformycin achieved was 0.4 to 0.5 p~ when the extracellular concentration was 1 p~. At 10 ~L M extracellular concentration, the intracellular concentration was 3-4 p~. Although deoxycoformycin at very low concentrations (1 or 10 FM) did not have any detectable effects on the growth of these cells, the nucleoside was found to be metabolized, and was phosphorylated to give the mono-, di-, and triphosphate derivatives. The triphosphate derivative was incorporated into cellular DNA with little incorporation into cellular RNA. Metabolism of deoxycoformycin in several mutant lymphoblastoid cells deficient in adenosine kinase and/or deoxycytidine kinase was found to be unchanged from wild-type cells, indicating that these major nucleoside kinases do not play a significant role in the phosphorylation of deoxycoformycin. These results may account, at least in part, for the differences that are observed between the pharmacologic inhibition of adenosine deaminase, and the inherited deficiency of adenosine deaminase. dCF' (Fig. 1, Miniprint) (1) is a potent inhibitor of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4). This nucleoside is an interesting and unique drug with wide clinical potential because it causes selective lysis of lymphocytes (desirable in lymphoproliferative diseases) or specific impairment of normal immune function which is not accompanied by cellular lysis (desirable in autoimmune diseases and graft versus host disease following organ transplantation). The * The first part of this study was presented in preliminary form at a workshop on deoxycoformycin held at the National Cancer Institute, April 1983. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Administration of high levels of dCF in humans results in 1) abnormally high ratios of dATP/ATP, 2) massive lymphocyte lysis, and 3) acute clinical toxicity (2)(3)(4)(5). Intracellular deoxynucleotide metabolism a t high dCF concentrations mimics that observed in children with hereditary deficiency of adenosine deaminase coupled with severe combined immunodeficiency disease (6,9). However, pharmacologic and disease patterns differ in that although intracellular ATP is severely depleted at high concentrations of the drug, ATP is not severely depleted in children with adenosine deaminase deficiency even after long-term therapy with erythrocyte infusion (6,7). Moreover, these children fail to exhibit any of the acute systemic toxicity observed at high dCF concentrations.
Administration of moderate doses of dCF in humans fails to cause 1) alteration in dATP/ATP ratios in lymphocytes, 2) acute clinical toxicity or 3) rapid lymphocyte lysis (3, 8). However, its ability to impair the immune system is retained. When low concentrations of dCF are incubated with human lymphocytes, both antibody-dependent and non-antibody-dependent cellular cytotoxicity are irreversibly impaired with no alteration in dATP/ATP (4). The lymphotoxic action of dCF under conditions where dATP/ATP pools are not altered suggests that a mechanism may exist which does not involve perturbed nucleotide pools or inhibition of adenosine deaminase per se, but metabolized products of dCF itself.
Deoxycoformycin has heretofore been considered a relatively inert nucleoside analogue which binds principally to adenosine deaminase. The possibility that dCF might be used as a substrate by any normal cellular pathways has not been widely investigated. However, previous studies have shown that dCF is not a substrate for purine nucleoside phosphorylase, a major degradative enzyme of purine salvage (10). As a consequence, it does not seem likely that this nucleoside analogue enters the major degradative pathways for purines. Therefore, it seemed logical to us to explore anabolic pathways of phosphorylation. In this context, one study has shown that dCF can be phosphorylated by L1210 cells in vitro (11).
However, in this study only the monophosphate form was detected.
Participation in major anabolic pathways requires formation of the triphosphorylated derivative of dCF. We therefore examined two cell lines treated with dCF for the presence of higher phosphorylated forms. After modifying standard procedures of nucleotide extraction to take into account the lability of these compounds, we did indeed find significant levels of mono-, di-, and triphosphorylated derivatives of dCF in lymphoblastoid cells. Moreover, we have evidence that dCF is incorporated into cellular DNA. These results support the hypothesis that the action of dCF is not limited to inhibition of adenosine deaminase.

EXPERIMENTAL PROCEDURES AND RESULTS'
Dose Response to Deoxycoformycin-The response of CEM and M o b 4 cells to dCF was compared with the response of these cells to the growth-limiting nucleosides, a d o , Ado, and araA. The effects of each of the nucleosides on cell growth was not readily apparent until 72 h. At that time, dCF was far less cytotoxic than Ado, &do, or araA. The concentrations at which there was 50% inhibition of cell growth were 3 X M (dAdo), 1 x M (Ado), and >1.0 x 10P M (dCF). A substantial contributor to a lack of cytotoxicity of the nucleosides is intracellular deamination which occurs via adenosine deaminase. As has been shown by others, when adenosine deaminase was inhibited by dCF (10 pM), dAdo (10 p~) and araA (10 p~) were particularly toxic to cells (see Fig. 2 Miniprint). When incubated with 10 ~L M dCF alone, these cells did not exhibit any significant changes in their intracellular dATP levels. However, cells incubated with 100 p~ dAdo in the presence of 10 p~ dCF were found to accumulate large amounts of dATP in their cells. After 24-h incubation at 37 "C, the amount of dATP in CEM and M o b 4 cells incubated with 100 pM dAdo in the presence of 10 p~ dCF was found to be around 614 pmol/106 cells and 460 pmol/106 cells, respectively. Lower levels of dATP accumulate in cells that were incubated with 100 pM dAdo alone. This was really not surprising since it is known that dAdo undergoes rapid deamination by adenosine deaminase to form dIno in the absence of any adenosine deaminase inhibitors. This experiment serves to demonstrate that, when its deamination is inhibited by dCF, dAdo can be phosphorylated to form dATP. Cellular Uptake of Deoxycoformycin-CEM and Molt-4 cells were found to exhibit similar kinetics in their uptake of dCF with respect to time (Fig. 3). It was found that the uptake of dCF initially proceeded rapidly and was linear for about an hour before slowly leveling off until a steady intracellular level was reached after about 10 h. At 1 p~ extracellular concentration, the level reached after 24 h for CEM and Molt-4 cells was 1.5 pmol/106 cells and 2.0 pmol/lO" cells, respectively. At 10 p~ extracellular concentration, this level was 14 pmol/106 cells and 16 pmol/106 cells, respectively. The intracellular concentration of dCF that was taken up in these cells (assuming a volume of 4.1 p1/106 cells) would be 0.4 to 0.5 p~ when the extracellular concentration was 1 p~. At 10 p~ extracellular concentration, the intracellular concentration was between 3 and 4 p~. In contrast, the uptake of the adenosine analog, araA, or the natural nucleoside, a d o , was found to be more rapid, with higher saturation levels being reached at  At high concentrations much earlier times. The level of araA or dAdo taken up into these cells was 100-fold higher (intracellular concentration of 400-500 p~ when the extracellular nucleosides were 10 p~) than that for dCF, undoubtedly due to the fact that araA and dAdo are rapidly metabolized inside the cell and the experiments described here reflect both uptake and metabolism. The concentration dependence of the uptake of dCF into CEM and Molt-4 cells was examined at various extracellular concentrations ranging from 1 to 500 p~. The uptake of dCF was found to be a biphasic process. Deoxycoformycin was taken into the cells in a linear fashion only at very low concentrations (up to 50 p~) , but slowly began to level off at higher concentrations. From the double reciprocal plots of 1/ u ( u is picomoles/106 cells/min) uersui l/[dCF] (mM-I), it was apparent that there were two (additive) uptake mechanisms, one operating more efficiently at low concentrations, and the other at high concentrations. The apparent VmaX and K, values associated with these two mechanisms for CEM and Molt-4 cells are listed in Table 11.
Metabolic Fate of Deoxycoformycin-Experiments to investigate the metabolic fate of dCF in in vitro cultures of CEM and Molt-4 cells were carried out by incubating cells with radioactive dCF and isolating labeled nucleotides using HPLC on an anion-exchange column.
A representative HPLC chromatogram of the absorbance at 254 nm of extracted nucleotides from CEM or Molt-4 cells incubated with [3H]dCF is shown in Fig. 44. At the bottom of Fig. 4 are profiles of the radioactivity associated with the various absorbance peaks. A major peak, corresponding to that of authentic dCF, was seen at about 2 min elution time. Three other major peaks were seen at 10, 24, and 43 min elution time. These radioactive peaks were representative of those peaks we predicted for the mono-, di-, and triphosphate derivatives of dCF. Five to seven other minor peaks were also seen. From the results of seven experiments, it was apparent that there was some variability in the distribution of radioactivity that was associated with each of the peaks (Table  111). The peak containing unmodified [3H]dCF (peak I) varied from as low as 32.1% (in Experiment 4) to as high as 92.3% (in Experiment 2) of total radioactivity for CEM cells. With M o b 4 cells, [3H]dCF extracted varied from as low as 22.6% (in Experiment 3) to as high as 94.9% (in Experiment 2). It appeared likely that the variation in amount of dCF present in phosphorylated forms was partially reflective of the instability of the compounds. When the isolation, extraction, and identification of nucleotides was carried out rapidly, most of the intracellular dCF was converted to phosphorylated forms. Four major peaks (peaks I, IV, VII, and IX) were always detected, along with five to seven other minor peaks. We postulated, based on their retention times, that peaks I, IV, VII, and IX represented dCF, dCFMP, dCFDP, and dCFTP. The identities of the minor peaks were not known, but they may represent the phosphorylated derivatives of the ringopened or degraded nucleoside. However, confirmation was  a Peaks denoted by double asterisks (**) are those identified to be deoxycoformycin (peak I), and its mono-(peak IV), di-(peak VII), and triphosphorylated derivatives (peak IX).
Percentage calculations are based on the total radioactivity in these 11 peaks.
Elution time is the mean of values from these seven experiments.
hampered by the lack of authentic standards of dCFMP, dCFDP, and dCFTP. T o confirm peak identities, a portion of the nucleotides extracted from cells was treated with alkaline phosphatase to degrade all the nucleotides to nucleosides, which were subsequently fractionated on a pBondapak C,, (reverse-phase) column. Degradation of all the putative dCF nucleotides resulted in only two peaks (Fig. 4B). The top part shows absorbance at 254 nm and the bottom shows radioactivity present in the same fractions. The two radioactive peaks were thought to represent those of dCF (peak 2) and a breakdown product of dCF (peak 1).
Confirmatory tests were also done by performing enzymatic shift reactions with nucleosides in peaks 1 and 2 using adenosine deaminase and purine nucleoside phosphorylase. Authentic [3H]dCF was also subjected to adenosine deaminase and purine nucleoside phosphorylase treatment, under the same experimental conditions as that used for the CEM and Molt-4 samples. Authentic [3H]dCF, when treated under these conditions, gave rise to the same two peaks (peaks 1 and 2) observed on the C,, column after alkaline phosphatase treatment of the CEM and Molt-4 samples (Fig. 5A). Peak 2 had UV absorbance at 254 nm, and was that due to intact dCF. However, peak 1 did not have any detectable UV absorbance at 254 nm, and we have postulated that it represents a breakdown product of dCF that may result from an opening of the seven-membered ring structure. It was noted that greater than 95% of the radioactivity in authentic, intact [3H] dCF which was not subjected to enzyme treatment fractionated in peak 2, with the remainder in peak 1 (data not shown). The ratio of radioactivity in peak 1 to peak 2 increased with time in samples of authentic [3H]dCF, probably due to degradation of the highly unstable compound under low pH conditions. These experiments also confirmed that authentic dCF was not a substrate for adenosine deaminase or purine nucleoside phosphorylase (Fig. 5A). Peaks 1 and 2 isolated from CEM and M o b 4 cells were also not altered by adenosine deaminase or purine nucleoside phosphorylase (Fig. 5, B and C, respectively). Based on the results of these experiments,  we concluded that peaks 1 and 2 represent a breakdown product of dCF and authentic dCF, respectively. Further confirmatory tests were also carried out by similarly treating each of the four major peaks (I, IV, VII, and IX) isolated from HPLC (as described in Table 111) with alkaline phosphatase, adenosine deaminase and purine nucleoside phosphorylase. After treatment with alkaline phosphatase, peaks I, IV, VII, and IX gave rise to peaks 1 and 2 on the CIS column. Once again, adenosine deaminase and purine nucleoside phosphorylase treatment did not alter peaks 1 and 2, indicating that they represent the breakdown product of dCF and authentic dCF, respectively. Thus, these experiments substantiate our postulation that peaks I, IV, VII, and IX from the Partisil-10 SAX column represented dCF, dCFMP, dCFDP, and dCFTP, respectively. Table IV is a summary of the distribution of radioactivity among the dCF, dCFMP, dCFDP, dCFTP, and other minor peaks.
Metabolic Fate of Deoxycoformycin in Kinase Mutant CEM Cells-In human T lymphoblastoid cells, nucleosides are converted to nucleoside monophosphates by several nucleoside kinases of variable specificities. When it became apparent that dCF was metabolized in lymphoblastoid cells, we sought to determine whether dCF was phosphorylated by the same enzymes which are known to convert deoxyadenosine to deox-yadenosine monophosphate in cells. Adenosine kinase and deoxycytidine kinase have both been shown to phosphorylate deoxyadenosine (12,13). To investigate the possible roles of these two enzymes in dCF phosphorylation, four mutant CEM cell lines (kindly provided by Dr. Buddy Ullman, University of Kentucky) were cultured in the presence of [3H]dCF. Each of the four cell lines was deficient in hypoxanthine-guanine phosphoribosyl transferase.
The results are depicted in Fig. 6 (A-D). In A, the cell line was totally deficient in adenosine kinase activity (BU-CEM-Tub4-M10-2) and in B, cells were deficient in deoxycytidine kinase activity (BU-CEM-AraC-8D). In neither mutant, was the phosphorylation pattern of dCF altered from the original CEM line. In C, the double mutant deficient in both adenosine kinase and deoxycytidine kinase (BU-CEM-AraC-8D-MMPR-10-5) was tested. As in the earlier experiment, an identical pattern of dCF phosphorylation was obtained. Finally, the CEM line containing only the hypoxanthine-guanine phosphoribosyl transferase deficiency (BU-CEM-HGPRT-) was utilized and shown to be identical to wild-type CEM cells.
From these data, we have concluded that neither Ado kinase nor dCyd kinase is responsible for dCF phosphorylation. Furthermore, dCF does not appear to compete with dAdo in enzymatic reactions which lead to the formation of monophosphate derivatives in the cells. The enzymes tested here for dCF phosphorylation do not represent an exhaustive list. Other candidates would include thymidine and uridine kinases, viral or mitochondrial deoxyribonucleoside kinases; however, these have not yet been tested. Studies in our laboratory with procaryotic cells suggest that a phosphotransferase efficiently converts dCF to its monophosphate derivative and a similar enzyme may be present in eucaryotic cells. 3 Incorporation of Deoxycoformycin into Cellular Nucleic Acids-Approximately 15-20% of the radioactivity in cells incubated with [3H]dCF was not extractable by 60% methanol. To account for this loss in radioactivity, we investigated the incorporation of dCF into cellular nucleic acids. CEM and Molt-4 cells were incubated with [3H]dCF for varying time periods. Following purification, the nucleic acids were treated with formamide, heated, and fractionated by CsS04 gradient centrifugation. DNA and RNA banded at densities between 1.42-1.48 g/ml and 1.62-1.68 g/ml, respectively. In control experiments, [3H]dCF was incubated with cellular nucleic acids which were subsequently carried through the same purification and fractionation experiments as those described above. Trichloroacetic acid-precipitable radioactivity was found only to be associated with DNA (or minor amounts of RNA) isolated from CEM or Molt-4 cells incubated with [3H] dCF (Fig. 7). The apparent amount of dCF incorporated into cellular DNA was 1-1.5 pmol/106 cells after 72 h (>go% of total radioactivity).
To investigate the nature of the tritium label that had been incorporated, total cellular nucleic acids, were degraded to their component nucleosides by DNase I, RNase A, bacterial alkaline phosphatase, and snake venom phosphodiesterase I. The nucleoside products were then analyzed by HPLC on a gBondapak CIS (reverse-phase) column. Degradation of total cellular nucleic acids gave rise to four radioactive peaks ( Fig.  8; peaks A-D). When the nucleosides in peaks A and B were incubated with adenosine deaminase or purine nucleoside phosphorylase, no change in the elution profile of either was observed. The material in peak B when incubated with pure adenosine deaminase efficiently inhibited the catalytic activ- ity of adenosine deaminase. These data indicate that peak B is authentic dCF and peak A is probably the breakdown product of dCF. The radioactive material in peaks C and D was also incubated with adenosine deaminase. Following incubation, the material in peak C co-eluted with dIno and the material in peak D co-eluted with Ino. The putative dIno and Ino compounds were incubated with PNP. Both compounds were converted to a new radioactive compound which coeluted with hypoxanthine. From these data, we were forced to conclude that the material in peak C was dAdo and the material in peak D was Ado. This conclusion was supported by the observation that peak D was not observed when DNA alone was degraded to its component nucleosides. Likewise, degradation of RNA alone did not yield peak C.
To examine whether tritium exchange was occurring at the nucleoside level (between dCF and Ado or a d o ) , we carried out a similar experiment with three cultures of CEM cells. Wild-type CEM cells, CEM cells containing the hypoxanthine-guanine phosphoribosyl transferase deficiency (BU-CEM-HGPRT-), and the double mutant deficient in both adenosine kinase and deoxycytidine kinase (BU-CEM-AraC-8D-MMPR-10-5) were cultured in the presence of [3H]dCF. Total cellular nucleic acids were extracted, degraded to component nucleosides and analyzed as above by HPLC. In each sample we observed a similar pattern; radioactive material co-eluted with authentic dCF and the breakdown product of dCF as well as in a broad peak at the position of adenosine and deoxyadenosine. When the material from the Ado and dAdo peaks was incubated sequentially with adenosine deaminase and purine nucleoside phosphorylase, we obtained the expected products, inosine, deoxyinosine, and finally hypoxanthine. The only variation we observed among the three cell lines was that the overall incorporation of radioactive material into DNA was lower in the cell line containing the double mutant for kinase deficiency than in the parent lines, but we believe this is attributable to the fact that the mutant cell line had a relatively slower growth rate than the parent cell lines. If tritium exchange were occurring at the nucleoside level, we would have predicted that no radioactive Ado or dAdo would be detected in the nucleosides generated from cellular nucleic acids.
Thus, the mechanism of intracellular conversion of the tritium label from dCF to Ado or dAdo is not readily discernible. The commercial preparations of [3H]dCF we used in these experiments do not contain detectable levels of Ado or dAdo (limit of detection was <0.02%). The data from the mutant cell lines coupled with the absence of radioactive phosphorylated derivatives of Ado or dAdo in intracellular extracts indicates that the label transfer to Ado or dAdo may occur when the dCF is incorporated into DNA.   conditions are used (14). Thus, the level of incorporation of dCF into nucleic acids was considerably lower, 1-2 pmol/106 cells, than that observed for other nucleosides. However, when the intracellular concentration of dCF achieved in cells is considered, 4 pM versus the intracellular concentration achieved for araA or dAdo, 400-500 p~, the incorporation of dCF into nucleic acids was relatively efficient.

DISCUSSION
Deoxycoformycin is a transition-state enzyme inhibitor which has attracted clinical interest because it is a selective immunosuppressant agent. When administered to patients, dCF precipitates a series of biochemical events that result in selective lymphocyte lysis (at high drug concentration) or impairment of immune function without lysis (at low drug concentration). Heretofore, the action of dCF was thought to result solely from its inhibition of the catalytic activity of adenosine deaminase.
In this study, we have shown for the first time that dCF entered the major nucleoside anabolic pathways in lymphoblastoid cells. We attributed the inability of others to demonstrate significant metabolism of dCF to two factors: 1) low specific activity radiolabeled dCF, and 2) instability of the phosphorylated derivatives of dCF to low pH extraction conditions. The problem of low specific activity was addressed satisfactorily when we obtained a uniformly labeled dCF from Moravek Biochemicals which was 5 times the specific activity of previous preparations of dCF. All nucleotide extractions were carried out in 60% methanol, accompanied by rapid sample processing to solve the experimental problem of nucleotide instability. The substantial variations we observed among experiments in recovery of dCF nucleotides was directly related to the length of time between sample extraction and analysis. Thus, with modified procedures, dCF was shown to be converted to mono-, di-, and triphosphorylated derivatives and ultimately incorporated into DNA and, to a far lesser extent, into RNA. The first step in the process, conversion to the monophosphate nucleoside, did not occur via the enzymes Ado kinase and dCyd kinase. Thus, dCF did not compete in kinase reactions with nucleosides adenosine and deoxyadenosine that accumulate when ADA is inhibited. When dCF was present as the triphosphorylated derivative, it functioned as a fairly efficient substrate for an as yet unidentified nucleotide-polymerizing activity and was incorporated into DNA as demonstrated by recovery of intact dCF following hydrolysis of nucleic acids.
When total nucleic acids were hydrolyzed, we were able to isolate not only radiolabeled dCF and its ring-opened structure but also radiolabeled dAdo and Ado. These natural nucleosides appeared to be incorporated into DNA and RNA,

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Metabolism of Deoxycoformycin in Human Lymphoblastoid Cells since they were not isolated in control experiments in which labeled dCF was incubated with DNA and carried through the isolation and fractionation procedures. The conversion of radiolabel from dCF to Ado and dAdo did not appear to occur metabolically, as in no instance were radiolabeled nucleotides of Ado or dAdo isolated from cells incubated with dCF. Isotope exchange from dCF to Ado and dAdo specifically (but not other nucleotides) did not appear to be a feasible mechanism, but we cannot completely rule out this possibility. However, when radioactive dCF was incubated with cells deficient in both adenosine kinase and deoxyadenosine kinase and nucleosides were generated from cellular nucleic acids, we observed a similar pattern with radioactivity associated with Ado and &do. This experiment seems to indicate that isotope exchange is not occurring a t the nucleoside level. Other potential mechanisms for exchange of radiolabel involve the DNA itself by close positioning of dCF and dAdo monomers, or by a DNA repair enzyme which may be capable of converting the dCF ring structure to dAdo.
The existence of phosphorylated derivatives of dCF and incorporation of these derivatives into cellular DNA raises questions about the mechanism of action of the drug. Since dCF has been shown here to be converted to nucleotide forms, it is possible that these compounds inhibit multiple enzymes.
As an example, dCF monophosphate, enzymatically synthesized from dCF using a preparation of 5'-nucleoside phosphotransferase from Serratia marcescens (15), has been shown by Frieden et al. (16) to be a competitive inhibitor of AMP deaminase with a K, of lo-'' M. Concentrations of dCF monophosphate in excess of this K, (3 X M), are easily achievable, as shown herein, in lymphoblasts at an extracellular dCF concentration of 10 pM.
Recently, two groups have observed abrupt phenotypic shifts in residual leukemic cells in certain patients treated with high levels of dCF. Murphy et al. (17) have reported that during the course of a 1-month treatment with dCF, a child with typical acute lymphoblastic leukemia quite suddenly experienced total marrow replacement of lymphoblasts showing T-cell characteristics with myeloblasts. Similarly, Hershfield et al. (18) have reported that in a single patient treated aggressively with dCF, there was a rapid conversion of leukemic cells from lymphoblast morphology to a typical promyeloblastic morphology. The potential role of dCF or any of its metabolites in precipitating phenotypic shifts in multipotent hematopoietic stem cells is unknown, but it is reasonable to consider that phosphorylated derivatives of dCF may have multiple effects on the anabolic pathways in cells which are exposed to the drug for prolonged intervals. These effects may to 1.8 X include unusual organ toxicities which have been associated with the use of high levels of this drug (3) as well as profound alterations in cellular morphology that may be a reflection of changes in gene expression in hematopoietic cells.  Pronase was from i a l a v " n n n g corp. (Sa" olego, CAI.

Metabolism of Deoxycoformycin in Human Lymphoblastoid Cells
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