Chlorambucil pharmacokinetics and DNA binding in chronic lymphocytic leukemia lymphocytes.

Chlorambucil (CLB) uptake by chronic lymphocytic leukemia lymphocytes was studied using a radiometric and a newly developed high-performance liquid chromatography assay. CLB labeled with 14C in either the chloroethyl group or phenyl ring was used with identical results. Drug accumulation by the cells was found to peak at 30 s, was independent of temperature, and was proportional to medium CLB concentration over a wide range. Efflux from cells loaded with CLB and resuspended in drug-free medium was nearly complete at 30 s. The metabolic inhibitors 2-deoxyglucose and NaN3, the nitrogen mustard transport inhibitor hemicholinium-3, and another alkylating agent, melphalan, had no effect on drug uptake. We conclude that CLB enters and exits chronic lymphocytic leukemia lymphocytes by simple diffusion. Cells from 17 patients with all stages of chronic lymphocytic leukemia were studied including three with CLB-resistant disease, and no heterogeneity was found in the peak cell-associated CLB content or in metabolite pattern on high-performance liquid chromatography. These findings make it unlikely that transport or cellular drug metabolism are factors in drug resistance. Drug-DNA binding was found to be temperature-sensitive and increased with time of incubation. Gel filtration of DNA before and after enzymatic digestion indicated the presence of drug-DNA adducts. High-performance liquid chromatography analysis of digested DNA and DNA treated by neutral thermal hydrolysis suggested the presence of multiple adducts. Most of the radioactivity was found as purine adducts. Studies with CLB labeled at two different sites revealed the presence of the phenyl group and ethyl chains in the adducts. A survey of patients showed increased drug-DNA binding in cells from patients with clinical CLB resistance.

Comparison of lipid composition and 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization measurements of hairy cells with monocytes and lymphocytes from normal subjects and patients with chronic lymphocytic leukemia.

In this report, we compare the lipid composition and fluorescence polarization properties of hairy cells with those of monocytes and lymphocytes from normal subjects and of lymphocytes from patients with chronic lymphocytic leukemia. For hairy cells, the cholesterol content was 4.66 +/- 1.49 (S.D.) mumol/10(9) cells, and the cholesterol/phospholipid ratio was 0.60 +/- 0.09. These were significantly higher than the values of normal lymphocytes, (cholesterol content, 2.75 +/- 0.65 mumol; cholesterol/phospholipid ratio, 0.50 +/- 0.07) or of chronic lymphocytic leukemia lymphocytes (cholesterol content, 1.76 +/- 0.43 mumol; cholesterol/phospholipid ratio, 0.44 +/- 0.07). Normal monocyte values (cholesterol content, 5.81 +/- 2.08 mumol; cholesterol/phospholipid ratio, 0.59 +/- 0.06) were similar to those of hairy cells. Using the probe 1,6-diphenyl-1,3,5-hexatriene, the fluorescence polarization value at 25 degrees for hairy cells was 0.302, compared to the value of 0.259 obtained with chronic lymphocytic leukemia lymphocytes. Intermediate values (0.294) were obtained with normal lymphocytes and monocytes. Fluorescence polarization values were higher in hairy cell membranes than in chronic lymphocytic leukemia lymphocyte membranes, indicating a low fluidity in the former cell, compatible with their higher cholesterol content and cholesterol/phospholipid ratio. These studies show that two neoplastic cells, hairy cells and chronic lymphocytic leukemia lymphocytes, differ markedly in membrane fluidity and that a high membrane fluidity does not necessarily occur in neoplasia.

Relationship of dehydroascorbic acid transport to cell lineage in lymphocytes from normal subjects and patients with chronic lymphocytic leukemia.

Dehydroascorbic acid is the principal form for the cellular uptake by blood cells of vitamin C. Since previous studies from this laboratory had shown a higher content of ascorbic acid and dehydroascorbic acid (DHA) in chronic lymphocytic leukemia (CLL) lymphocytes when compared to their normal counterparts, DHA uptake was characterized using these cells. The affinities of CLL and normal lymphocytes for DHA uptake were similar, as demonstrated by the Km values of 3.7 and 3.5 mM, respectively. Differences were found in other kinetic constants of DHA uptake. The Vmax for normal lymphocytes, 634 mumol/liter cell H2O/min, was approximately twice that of CLL cells, 392 mumol/liter cell H2O/min. In addition, the initial velocity and the maximal DHA uptake by normal lymphocytes were greater than that of CLL lymphocytes. These differences were not simply a reflection of lymphocyte subsets since CLL B-cells demonstrated lower uptake rates than did normal B-cells whereas CLL T-cells were similar to their normal counterparts. The alterations appear to be specific for the leukemic B-cell since they were not shared by neoplastic cells from two patients with T-cell CLL. When analyzed in light of the 3-fold greater cellular DHA and ascorbic acid content in B-cell CLL as compared to normal lymphocytes, these kinetic parameters support the occurrence of a concentration-dependent transport system for DHA. We conclude that the DHA uptake properties of CLL lymphocytes of B-cell origin serves to distinguish this lineage from T-cell CLL or normal lymphocytes.

Purification and characterization of actin from normal and chronic lymphocytic leukemia lymphocytes.

Previous studies from this laboratory have shown actin to be a major protein of human lymphocytes (Stark, R., Liebes, L. F., Nevrla, D., and Silber, R. Biochem. Med., 27: 200-206, 1982). We now report the purification to homogeneity and characterization of actin from blood lymphocytes of normal subjects and patients with chronic lymphocytic leukemia. The recovery of the purified protein was about 20%. The properties of the lymphocyte actins were compared to each other and to those of rabbit skeletal muscle actin. Lymphocyte actin consisted of beta and gamma forms in a 2:1 ratio. The Mr 42,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Normal and leukemic lymphocyte actin had similar polymerization properties as assessed by viscosity measurements at 25 degrees and 4 degrees, and the ultrastructural appearance of the filaments was the same. Similar patterns were observed between normal and chronic lymphocytic leukemia actin tryptic digests analyzed by high-performance liquid chromatography. The Vmax of the actin-activated myosin Mg2+ ATPase activity was compared using rabbit skeletal muscle heavy meromyosin and subfragment 1 preparations. The values obtained with rabbit skeletal muscle and normal lymphocyte actin were identical. The Vmax observed with chronic lymphocytic leukemia lymphocyte actin was 70% of that obtained with normal lymphocyte actin. The amount of actin needed to produce half-maximal activation (Kapparent) of heavy meromyosin and subfragment 1 were, respectively, 26 and 25 microM for normal lymphocytes and 18 and 24 microM for chronic lymphocytic leukemia lymphocytes. The anomalous ATP activation by actin did not reflect differences in B-:T-cell subpopulations between chronic lymphocytic leukemia and normal lymphocytes. The possible significance of the observed differences between the myosin Mg2+ ATPase activation by chronic lymphocytic leukemia and normal lymphocyte actin is discussed.

Ribonucleotide content of mononuclear cells from normal subjects and patients with chronic lymphocytic leukemia: increased nicotinamide adenine dinucleotide concentration in chronic lymphocytic leukemia lymphocytes.

The ribonucleotide content of lymphocytes obtained from normal subjects and patients with chronic lymphocytic leukemia (CLL) was determined by means of high-performance liquid chromatography. The levels of normal B- and T-cells were compared to each other as well as those of their CLL counterparts. Unfractionated CLL lymphocytes, predominantly B-cells, had significantly lower levels of adenosine-5'-triphosphate, cytidine-5'-triphosphate, uridine-5'-triphosphate, cytidine-5'-diphosphate, and guanosine-5'-phosphate, while the concentration of nicotinamide-adenine dinucleotide was significantly higher than in normal unfractionated lymphocytes which consisted mainly of T-cells. For enriched populations: (a) CLL B-cells had much lower adenosine-5'-triphosphate (3439 versus 5689) (pmol/1 X 10(7) cells), cytidine-5'-triphosphate (107 versus 313), guanosine-5'-triphosphate (462 versus 978), and uridine-5'-triphosphate (633 versus 1214) than normal B-cells; (b) CLL T-enriched subpopulations had significantly lower ribonucleoside triphosphates, adenosine-5'-triphosphate (3217 versus 5468), cytidine-5'-triphosphate (119 versus 209), guanosine-5'-triphosphate (422 versus 826), and uridine-5'-triphosphate (504 versus 969) than normal T-cells. The lower ribonucleoside triphosphate levels found in unfractionated CLL lymphocytes, therefore, are the result of differences between the CLL and normal B-cells as well as between CLL and normal T-cells. These findings establish a framework for studying the reasons underlying the decreased ribonucleoside triphosphate levels in unfractionated CLL lymphocytes. T-helper and T-suppressor lymphocytes showed similar ribonucleotide patterns. Nucleoside and base levels were significantly higher in normal monocytes than in normal lymphocytes. The only compound found to be increased in the CLL B-lymphocytes when compared to their normal counterparts was nicotinamide-adenine dinucleotide. The level in CLL lymphocytes was 404 versus 209 pmol/10(7) cells for normal B-lymphocytes. No correlation was found between any ribonucleotide levels and the expression of 5'-nucleotidase activity.

Phase I trial of escalating dose doxorubicin administered concurrently with alpha 2-interferon.

The clinical use of alpha 2-interferon and doxorubicin is based on in vitro and preclinical in vivo observations of synergistic antitumor efficacy. To test this combination a Phase I clinical and pharmacokinetic study of the concurrent use of alpha 2-interferon and doxorubicin was initiated in patients with malignant solid tumors. Each 5-wk treatment cycle consisted of 3 wk of drug administration and 2 wk of rest. The alpha 2-interferon was administered s.c. at a constant dose of 10 million IU/m2 on Mondays, Wednesdays, and Fridays in all patients while the doxorubicin was administered weekly beginning with a dose of 5 mg/m2 and escalated to the maximum tolerated dose of 25 mg/m2. At least three evaluable patients were entered at each dose level, and no dose escalations were allowed within patients. The dose-limiting toxicities were granulocytopenia and thrombocytopenia. Hepatic enzyme elevations and systemic symptoms due to interferon occurred at all dose levels. None was severe or dose limiting, and all were reversible. These toxicity data suggest that the hepatotoxic effects of interferon do not enhance doxorubicin toxicity when given by this dose and schedule. Doxorubicin plasma levels were measured at each dose level. The recommended dose of doxorubicin is 25 mg/m2 per wk when administered with 10 million IU/m2 of interferon in this schedule. This schedule allows for the administration of a greater total dose of doxorubicin than has been achieved when given every 3 wk with the same dose and schedule of alpha 2-interferon in a parallel study.

A reappraisal of leukocyte dehydroascorbate reductase.

Dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1) activity was determined in human leukocyte homogenates using a direct spectrophotometric assay. Despite previous studies, using a less sensitive coupled assay, which reported that this enzyme was present in leukocytes, we found that neither neutrophil nor chronic lymphocytic leukemia lymphocyte extracts had detectable activity. Furthermore, when the product was quantitated by HPLC, protein-dependent generation could not be demonstrated. Mixing experiments with a partially purified enzyme preparation from spinach leaves provided no evidence for the presence of an inhibitor in neutrophil homogenates. These findings suggest that in human leukocytes, dehydroascorbate reduction does not occur enzymatically.

The solution behavior of the bovine myelin basic protein in the presence of anionic ligands. Binding behavior with the red component of trypan blue and sodium dodecyl sulfate.

The interaction of the azo dye (2,3'-dimethyldiphenyl-7-azo-8-amino-1-napthol 3,6-disulfonic acid (TBR) and sodium dodecyl sulfate with the bovine myelin basic protein has been studied using absorbance, circular dichroism and 220 MHz PMR spectroscopy. Additional analyses of the binding reaction were carried out using light scattering, ultracentrifugal and electrophoretic techniques. A procedure for preparing pure TBR was developed. A modified structure for this synthesized TBR has been suggested. The mechanism of TBR binding to the myelin basic protein was found to be metachromatic. In addition, the interaction of TBR with the basic protein which gives rise to aggregation of the dye bound species was found to be analogous to the model proposed by Schwarz, G. and Seelig-Löffler, A. ((1975) Biochim. Biophys. Acta 379, 125-138) to explain the binding of acridine orange with poly (alpha-L-glutamic acid). PMR spectral analyses suggested that arginine residues provide the majority of primary sites of attachment on the basic protein for TBR. The effect of sodium dodecyl sulfate binding with the bovine myelin basic protein was found to induce a minimal change in the conformation of the protein. The induction of only about 20% alpha helial structure could be demonstrated and the binding was reversed by raising the solution temperature to 73 degrees C. The difference in the observed behavior of basic protein arising from TBR binding as opposed to the binding of sodium dodecyl sulfate is viewed as resulting from two different binding mechanisms. The binding behavior of TBR is primarily a consequence of charge-charge interaction while the binding effects of sodium dodecyl sulfate are a consequence of hydrophobic interaction. The sodium dodecyl sulfate binding acts as a shield which limits charge-charge interaction in the basic protein molecule thus preventing aggregate formation while TBR imposes no such restraints.

Solution behavior, circular dichroism and 22 HMz PMR studies of the bovine myelin basic protein.

Bovine myelin basic protein has been investigated with regard to its solution behavior, circular dichroism and 220 MHz PMR spectral properties. At pH 4.8 gamma/2=0.1 acetate buffer, light scattering yielded a Mr of 17 700 and a virial coefficient of 1.0-10(-4) mol-ml/g2. Above pH 7.0 the protein was found to aggregate to higher mol. wt species. Sedimentation experiments at pH 4.8 yielded s degrees 20,w of 1.27 S at gamma/2=0.1 and 1.46 S at gamma/2=0.35. The diffusion coefficient determined from ultracentrifugal experiments was 7.25-10(-7) cm2/s at gamma/2=0.1 and 0.35. The value of f/f0 from diffusion at pH 4.8 and gamma/2=0.35 was 1.64, corresponding to an axial ratio of 11 to 1. The radius of gyration was calculated as 4.28 nm and the root mean square end to end distance was 10.5 nm. At pH 9.0, gamma/2=0.1, s degrees 20,w was 1.71 S and D degrees 20,w was estimated at 7.4-10(-7) cm2/s. The behavior at pH 9.0 reverted to the behavior at pH 4.8 when the pH was readjusted. The E1%/1cm=5.64 at 276.4 nm and 225 at 196 nm. Titration of the protein with trifluoroethanol elicited three distinct regions of conformation stability having increasing helical content as the mol fraction of trifluoroethanol increased. The results of the present study have permitted some comparison of analogous properties and conformational behavior with the basic membrane protein cytochrome c.

Fluorescent labeling of DNA in solution with covalently bound acriflavin.

We have prepared a fluorescent derivative of DNA based on the acriflavin-Feulgen histological procedure for staining DNA. Our procedure involved binding acriflavin to DNA in solution by reacting the acriflavin with aldehydes formed on the deoxyribose of DNA by controlled removal of a few percent of the purine bases of the DNA. Partially depurinated DNA was reacted with the acriflavin reagent, and unbound acriflavin was removed by chromatography on Sephadex G-25 eluted with phosphate buffered guanidine -HCl. Such single-stranded depurinated DNA bound 0.36 acriflavin molecules per 100 purine bases per h of depurination. DNA containing one bound acriflavin per 200 bases reassociated at 85% of the value of control DNA. The acriflavin - DNA complex showed new absorption maxima at 466 and 370 nm. The fluorescent product had excitation maxima at 304 and 465 nm and an emission maximum at 502 nm. This labeling procedure should be useful in place of or in addition to radioactive labeling for DNA.

Human lymphocyte tubulin. Purification and characterization in normal and leukemic cells.

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119000. The protein was shown to consist of two subunits, with molecular weights of 61000 and 58000 comparable to the alpha and beta polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 +/- 0.86 . 10(6) M-1 and a level in normal lymphocytes of 1.21 . 10(2) +/- 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and Ka for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocytes tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity for function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.

Continuous-flow scanning of selected high-performance liquid chromatography peak components by microprocessor control. Application to analysis of extracts from human lymphocytes.

Continuous-flow wavelength scanning of compounds separated by high-performance liquid chromatography is achieved through the use of fixed and variable wavelength micro ultraviolet detectors connected in series but separated by a low-pressure three-way valve. Activation of the valve allows entrapment of selected peaks in the variable-wavelength detector without interfering with the response of the fixed-wavelength detector which is utilized for peak quantitation. A microprocessor program is employed to maintain control and accuracy during the scanning sequence. Good correlation was found between ultraviolet spectra of standards obtained on a conventional spectrometer and those on separated peaks. This system allows the identification and quantification of picomole amounts of peaks separated during one analysis of a biological sample.

Chlorambucil therapy in hairy cell leukemia: effects on lipid composition and lymphocyte subpopulations.

Two patients with progressive hairy cell leukemia following splenectomy were treated with low-dose daily chlorambucil. Both had an objective hematologic response as determined by a return to normal hematocrit and platelet count. This was also reflected in the mononuclear cell fraction by the normalization of cholesterol content, cholesterol/phospholipid ratio, and the lymphocyte subpopulations. This article confirms previous reports on the efficacy of chlorambucil in this setting and describes some morphological, and biochemical concomitant events.

Antioxidant enzymes in lymphocytes from normal subjects and patients with chronic lymphocytic leukaemia: increased glutathione peroxidase activity in CLL B lymphocytes.

The activities of several enzymes that protect against oxidative injury were determined in blood lymphocytes from patients with B chronic lymphocytic leukaemia (CLL) and from normal subjects. Similar glutathione reductase (GR), catalase and glucose-6-phosphate dehydrogenase (G6PD) activities were found in normal and CLL lymphocytes. Higher glutathione peroxidase (GP) activity was found in CLL lymphocytes. This activity in CLL B lymphocytes was 2-fold higher than that of normal B lymphocytes, and 3-fold higher than that of T lymphocytes from either source. Several disease processes have been associated with decreased glutathione peroxidase activity. Our finding with CLL B lymphocytes is believed to be the first example of an increased GP activity in a disease. It may reflect either the expansion of a rare type of B cell population or be an expression of the malignant process.

Relationship of fluorescence polarization to cell lineage in lymphocytes from normal subjects and patients with chronic lymphocytic leukemia.

Previous investigations have shown differences in fluorescence polarization between normal and chronic lymphocytic leukemia lymphocytes following incubation with the probe 1,6-diphenyl-1,3,5-hexatriene. In the present study, we determined the fluorescence polarization of unseparated or enriched subpopulations of T and B lymphocytes from normal subjects and patients with chronic lymphocytic leukemia. As had been observed by others, the mean polarization (P) value at 25 degrees C for unseparated chronic lymphocytic leukemia lymphocytes, .240 +/- .007 (N = 22), was lower than that of unseparated normal lymphocytes, .248 +/- .005 (N = 18), P less than .001 (Student's t-test). The difference was greater when B-enriched populations were compared. The mean P value of B-cell-enriched chronic lymphocytic leukemia lymphocytes, .240 +/- .007 (N = 5), was significantly lower than that of B-cell-enriched normal preparations, .256 +/- .004 (N = 5), P less than .001. In contrast, no significant difference was found between normal and chronic lymphocytic leukemia T cells. The anomalous fluorescence polarization manifested by chronic lymphocytic leukemia lymphocytes of B-cell origin serves to distinguish this lineage from its normal counterpart and from T cells of either source.

Decreased actin content of lymphocytes from patients with chronic lymphocytic leukemia.

Actin, a major cytoskeletal protein, was quantitated in normal and chronic lymphocytic leukemia lymphocytes. The actin content of normal human blood lymphocytes was 2.2 +/- 0.4 mg/10(9) cells and represented 6.6% +/- 1.8% of the total cellular protein. A significant decrease (p less than 0.001) was noted in chronic lymphocytic leukemia lymphocytes that contained 1.4 +/- 0.3 mg actin/10(9) cells, constituting 4.3% +/- 1.1% of the total protein. Normal T and B cells did not differ in actin content. Reduced actin levels were found in the T as well as in the B lymphocytes of "B-cell" chronic lymphocytic leukemia. The possible importance of the decreased actin level in the anomalous capping response and motility of chronic lymphocytic leukemia lymphocytes is discussed.

Electrophoretic mobility distributions distinguish hairy cells from other mononuclear blood cells and provide evidence for the heterogeneity of normal monocytes.

The electrophoretic mobility distributions of hairy cells, normal monocytes. CLL, and normal lymphocytes isolated from blood were determined by electrophoretic light scattering. Values obtained for hairy cells, 1.52 X 10(-4) cm2/V . sec, were indistinguishable from that of normal monocytes. The mobility of CLL lymphocytes was similar to that of normal B cells. After exposure to neuraminidase, hairy cells revealed a homogeneous distribution with a reduced mobility of 0.55 X 10(-4) cm2/V . sec, while normal monocytes showed a heterogeneous distribution of electrophoretic mobilities suggestive of subpopulations. The electrokinetic behavior of hairy cells thus differs from that or normal and CLL lymphocytes before, and from that of monocytes after, treatment with neuraminidase. The hairy cell therefore possesses a distinct pattern of surface charge properties that clearly distinguish it from the circulating B cells, T cells, or monocytes.

A spectrophotometric assay for dehydroascorbate reductase.

A simple spectrophotometric assay for dehydroascorbate reductase based on the change in absorbance associated with the formation of ascorbic acid is described. Using a partially purified preparation from spinach leaves, the reaction was found to be linear with time and enzyme concentration. The reaction rate determined by this assay correlated well with that obtained by a high-performance liquid chromatography method. Possible advantages over currently available assays as well as potential applications are discussed.

Human B lymphocytes show greater susceptibility to H2O2 toxicity than T lymphocytes.

Lymphocytes from patients with chronic lymphocytic leukemia (CLL) and from normal subjects were incubated with a glucose-glucose oxidase hydrogen peroxide (H2O2) generating system to study the effect of oxidant stress on these cells. Within 4 hr, 90% of normal but only 21% of CLL lymphocytes remained viable. When normal and CLL preparations enriched in B or T cells were exposed to H2O2, B lymphocytes from both groups were highly susceptible to oxidative damage while T lymphocytes were relatively resistant. The H2O2 scavenger catalase prevented the cytotoxicity. The present work identifies the human B lymphocyte as a cell that should be a suitable target for selective killing by H2O2-generating systems.