Nahed K. Ahmed

Non-transferrin-bound iron in long-term transfusion in children with congenital anemias

Non-transferrin-bound iron (NTBI), a potentially toxic compound, is increased in the serum of patients with iron overload and fully saturated transferrin. We found markedly elevated NTBI levels in 16 children (including nine with sickle cell disease and five with beta-thalassemia) with iron overload secondary to prolonged transfusion therapy. During iron chelation with subcutaneous desferrioxamine infusion, NTBI levels decreased to normal, but became elevated within 2 to 4 hours after discontinuation of desferrioxamine. NTBI causes hepatic and cardiac toxicity in experimental systems, but our patients lacked sufficient organ dysfunction for this association to be made. The use of continuous 24-hour chelation to maintain NTBI at low levels may prevent progressive iron toxicity in patients who first received chelation therapy at an older age or who already have evidence of cardiac damage.

Enzymes of the de novo and salvage pathways for pyrimidine biosynthesis in normal colon, colon carcinoma, and xenografts

Since current methods of chemotherapy for adenocarcinoma of the colon are essentially ineffective, this study was designed to test for enzymatic differences between tumors and normal colon that might form the basis for more effective treatment. Human colon tumor xenografts also were examined and were found to be very similar to primary tumors when tested for: uridine‐cytidine (Urd‐Cyd) kinase and orotidine 5′‐phosphate (OMP) decarboxylase activity, apparent Michaelis constants of Urd, ATP, and OMP, and temperature and pH optima for Urd‐Cyd kinase. However, enzyme activity levels varied from one xenograft line to another, and these differences could not be correlated with growth rate or sensitivity to 5‐flurouracil (5‐FU). The xenograft, therefore, may provide a suitable model for the study of human colorectal adenocarcinoma, but care must be taken to screen different lines in order to select ones that are comparable to primary tumors. Primary tumors and xenografts, when compared to normal colon, were found to have significantly higher specific activities of enzymes of both the de novo and salvage pathways of uridine monophosphate (UMP) biosynthesis. The activities of Urd‐Cyd kinase and OMP decarboxylase were greater by 132% and 91%, respectively, in primary tumors and 186% and 63%, respectively, in xenografts. Consequently, effective treatment of adenocarcinoma of the colon using inhibitors of pyrimidine nucleotide biosynthesis would probably require the combination of a compound that inhibits the salvage pathway, e.g., inhibitors of Urd‐Cyd kinase, with one that inhibits the de novo pathway, e.g., pyrazofurin or N‐(phosphonacetyl)‐L‐aspartate (PALA).

Nontransferrin-bound serum iron in thalassemia and sickle cell patients

Nontransferrin-bound iron (NTBI) was separated from transferrin bound iron (TBI) by DEAE-Sephadex-CDS filtration. TBI is eluted with Tris-NaCl buffer, NTBI that is retained on the column is eluted with citric acid. NTBI was identified in serum from thalassemia and sickle cell patients. Normal serum contained less than 6% NTBI as compared with 15-18% in patients sera. NTBI levels were decreased significantly after 8 hr chelation with deferoxamine (DFO).

Uptake and metabolism of daunorubicin by human myelocytic cells

SummaryDaunorubicin uptake and metabolism were studied in vitro with human myeloid leukemia cell lines (KG1, ML1); erythroleukemia cell line (K562); and myeloblasts from two untreated patients with acute myelogenous leukemia (AML). Uptake of daunorubicin by all the above was very similar, but metabolism of daunorubicin to daunorubicinol and the levels of reductase activity were extremely variable. We believe that this heterogeneity accurately reflects the in vivo situation in humans with acute leukemia. In vivo anthracyclines are subject to extensive metabolism, and the majority of patients do metabolize the drug to some extent; it is important, therefore, to use cell lines that reflect the in vivo metabolism. Conversely, rodent cell lines, which apparently lack one of the two major classes of daunorubicin reductase and do not appreciably metabolize daunorubicin, appear to be inadequate as models for studies designed to evaluate the enzymatic mechanisms of daunorubicin metabolism.

Phosphorylation of nucleosides catalyzed by a mammalian enzyme other than uridine-cytidine kinase

Abstract Cells of a 3-deazauridine-resistant line of cultured lymphoblastoid cells, RPMI 6410, were found to be essentially devoid of uridine-cytidine kinase activity. The resistant cells tolerated high concentrations of 3-deazauridine in culture, but retained sensitivity, relative to the parent cells, to 6-azauridine and 5-azacytidine, compounds previously recognized as substrates for uridine-cytidine kinase. Intracellular formation of the 5′-monophosphate esters of these analogs is a necessary step in expression of their cytotoxicity. The sensitivity of 3-deazauridine-resistant cells to 6-azauridine and 5-azacytidine is interpreted as indicating that phosphorylation of these analogs by some enzymatic route other than uridine-cytidine kinase may occur in these cells.

Comparative uptake, retention and cytotoxicity of daunorubicin by human myeloid cells

We studied the cellular uptake and retention of daunorubicin (D1) in two human leukemic cell lines (ML1 and K562) and myecloblasts from an untreated patient with acute myelogenous leukemia (AML). The rate of uptake and the steady-state level of D1 were not temperature dependent but increased markedly with increases in the pH of the medium. Also, saturation kinetics were not demonstrable using concentrations of D1 up to 111 microM. Together, these observation suggest a transport mechanism for D1 compatible with passive diffusion. Accumulation of D1 was increased only in cells from the AML patient with addition of sodium azide, whereas drug efflux was not increased significantly in the presence of glucose in MLI or K562 cells. Although the rate of uptake and steady-state levels of D1 were the same in these cells, metabolism and cytotoxicity of D1 differed. Our results indicate that ML1 cells can be used as a pharmacologic model for studying the metabolism and resistance of D1 in vivo.

Daunorubicin reductase activity in human normal lymphocytes, myeloblasts and leukemic cell lines

To exploit the full potential of daunorubicin chemotherapy, it is necessary to understand its metabolism. We have shown previously that daunorubicin reduction in human liver is mediated by both aldehyde and ketone reductases. This study shows that this is also the case in normal blood cells. However, myeloblasts from AML patients show different pH profiles from those observed for normal lymphocytes. Human myeloid cell lines (KG1, ML1 and K562) accurately reflect the reductase heterogeneity seen in AML patients. This is in contrast to L1210 and P388 murine cell lines, which do not readily metabolize daunorubicin. When studying daunorubicin metabolism, it is important to use only cell lines that metabolize the drug because daunorubicin is extensively metabolized to daunorubicinol in AML patients. The use of human rather than rodent cell lines may provide useful information to increase our understanding of the in vivo situation.

Multiple forms and inhibitors of uridine-cytidine kinase in neoplastic cells

1. Two forms (isozymes) of uridine (urd)-cytidine (cyd) kinase are present in the 30-50% ammonium sulfate fraction of the cytosols of L1210 ascites leukemia cells and a human malignant lymphoma. 2. These findings confirm those which described multiple forms of urd-cyd kinase in tumors with rapid growth rate. 3. Studied of inhibitors (nucleoside analogs) of urd-cyd kinase derived from L1210 and 6410 leukemia cells resulted in the finding of four possible inhibitors of this enzyme.

Contribution of drug transport and reductases to daunorubicin resistance in human myelocytic cells

SummaryWe developed three daunorubicin (D1)-resistant sublines (ML1/I, II, III) from the human myelocytic cell line (ML1). These sublines were 28-, 70- and 162-fold more resistant than sensitive (ML1/S) cells to the cytotoxicity of D1 and were cross-resistant to adriamycin, epiadriamycin, actinomycin D, VP-16, VM26, and mitoxantrone. Steady-state levels of D1 in resistant sublines I and II, in the presence or absence of azide, were not significantly different from those of sensitive cells. However, the steady-state level of D1 in subline III was significantly increased in the presence of sodium azide. D1 efflux was minimal in ML1/S and resistant cells in the absenced of glucose. Addition of glucose enhanced D1 efflux only in subline III. Verapamil increased the cellular levels of D1 and inhibited its efflux from resistant III cells but not from ML1/S cells. Verapamil also greatly enhanced the cytotoxicity of D1 for sublines I, II, and III. The differences between sensitive and resistant cells in D1 uptake and retention seemed inadequate to cause 162-fold resistance and suggested other factors may be contributing to the development of resistance. In support of this hypothesis, daunorubicin reductase activity was significantly lower in resistant cells than in ML1/S cells. The greatest decrease in activity occurred at pH 8.5 which represents aldehyde reductases. Currently, we are investigating other possibilities for D1 metabolism, such as aglycone and free radical formation.

Characterization of daunorubicin resistance in K562 leukemia cells lacking daunorubicin reductase activity

Daunorubicin (D1)-resistant cells have been isolated from daunorubicin reductase-deficient K562 cells, hence, metabolism of D1 to the alcohol metabolite daunorubicinol (D2) will not contribute to the development of resistance. The resistant cell lines were 22-123-fold resistant and were cross-resistant to a variety of drugs. Drug uptake and efflux were altered in the more resistant lines but not in the less resistant cells. Verapamil enhanced D1 cytotoxicity in all resistant lines; it inhibited D1 efflux in the higher resistant line thereby resulting in an increase in the cellular level of D1. However, this was not true for the less resistant line suggesting that verapamil enhancement of D1 toxicity in the less resistant line is probably due to other factors. Additionally, we have been unable to identify a marker glycoprotein in resistant cells. The changes observed in the resistant sublines are moderate and probably drug accumulation differences could not account for the degree of D1 resistance noted, nor could resistance be wholly reversed by calcium antagonist. Other factors may be involved in the development of resistance in these human cells.