Chemosensitivity of lymphocytes from patients with B-cell chronic lymphocytic leukemia to chlorambucil, fludarabine, and camptothecin analogs.

Chemosensitivity of B lymphocytes, obtained from 65 patients with B-cell chronic lymphocytic leukemia (B-CLL), Rai stages 0 through IV, was determined using the MTT assay. The results were expressed by the drug concentration required for 50% inhibition of cell viability (IC50). The cytotoxicity of chlorambucil (CLB) was compared with that of fludarabine and the DNA topoisomerase I inhibitors, camptothecin, 9-aminocamptothecin, 10,11-methylenedioxy-20(S)-camptothecin (10,11-MDC) and 9-amino-10,11-methylenedioxy-20(S)-campthothecin (9-A-10,11-MDC), and topotecan. Considerable heterogeneity in sensitivity to CLB was observed, with a median IC50 of 40.5 mumol/L in untreated patients. B-CLL cells from patients treated with CLB had a significantly higher median IC50 of 86.0 mumol/L (P < .01). Untreated as well as CLB-treated patients were divided into two subsets. For the purpose of this study, B-CLL lymphocytes with an IC50 CLB of less than 61.0 mumol/L were designated as "sensitive" and those with an IC50 CLB of > or = 61.0 mumol/L were designated as "resistant." After baseline assays, 15 untreated patients received CLB; after treatment, the IC50 increased in B-CLL lymphocytes from 13 of 15 patients. The response to CLB treatment, determined by its effect on the absolute lymphocyte count and by the Eastern Cooperative Oncology Group clinical criteria, was significantly better in patients whose lymphocytes had an IC50 CLB of less than 61.0 mumol/L before therapy (P < .01). B-CLL lymphocytes also had a variable degree of sensitivity in vitro to each of the other drugs. There was significant cross-resistance between CLB and fludarabine (P < 0.01). Whereas only 29% of CLB-resistant B-lymphocyte specimens obtained from individual patients were sensitive to fludarabine in vitro, 52% and 67% of CLB-resistant lymphocyte samples were sensitive to 10,11-MDC and 9-A-10,11-MDC, respectively. We have previously reported that p53 gene mutations were associated with aggressive B-CLL and a poor prognosis. B lymphocytes from seven patients with these mutations were resistant to CLB, and five of six were resistant to fludarabine. Lymphocytes from four of seven were resistant to 10,11-MDC, and three of four were resistant to 9-A-10,11-MDC. This study implies that the MTT assay may be useful in identifying subsets of CLL patients resistant to conventional chemotherapy. However, definitive conclusions can not be drawn in view of the small number of patients studied prospectively. In addition, these results suggest the potential of camptothecin-based therapy for patients unresponsive to standard treatment.

Glutathione depletion in chronic lymphocytic leukemia B lymphocytes.

Glutathione (GSH) content may be the major determinant of a cell's sensitivity to cytotoxic alkylating agents. In the present study, the GSH concentration was determined in lymphocytes isolated from the blood of normal subjects and patients with chronic lymphocytic leukemia (CLL). Comparable levels were found in both types of cells. Incubation for 20 hours led to a decrease in GSH to 51% of baseline values in CLL B cells. Under the same conditions, normal B- or T-lymphocyte GSH content remained constant. GSH depletion was shown to be a characteristic of the B-CLL B lymphocyte. It was not found in the T cells of patients with B-CLL or in cells from patients with T-CLL. Chlorambucil (CLB) contributes to the decrease in GSH in B-CLL lymphocytes; after incubation with the drug, lower levels of GSH were found than in the normal B or T lymphocytes, B-CLL T cells, or T-CLL (CD4 or CD8) cells. GSH depletion of CLL B lymphocytes may be related to the greater therapeutic efficacy of CLB in B-CLL than in T-CLL.

Effect of acetylpolyamines on in vitro protein synthesis and on the growth of a polyamine-requiring mutant of Escherichia coli.

The functions of acetylpolyamines were examined with respect to stimulation of protein synthesis and cell growth. Unlike polyamines, acetylpolyamines could not lower the optimal Mg2+ concentration of protein synthesis, and the degree of stimulation of protein synthesis by acetylpolyamines was small. The addition of N1-acetylspermine did not stimulate cell growth of a polyamine-requiring mutant of Escherichia coli MA261, although acetylspermine was accumulated in the cells. Acetylspermine did not interfere with polyamine stimulation of protein synthesis and cell growth of E. coli MA261. The binding of acetylpolyamines to RNA was very weak, and the binding of polyamines to RNA was not disturbed significantly by the presence of acetylpolyamines. When the growth of E. coli MA261 was stimulated by addition of polyamines, significant amounts of acetylpolyamines were also formed in the cells. These results suggest that acetylation of polyamines, together with polyamine excretion, may regulate the intracellular level of the parent polyamines when excess amounts of polyamines accumulate intracellularly.

Diacetylputrescine and its analog suppress c-myc expression and activation of human B-lymphocytes.

Our studies demonstrate that diacetylputrescine (TMBA), and its analog hexamethylenebisacetamide, HMBA, inhibit the expression of the c-myc oncogene in activated human B-lymphocytes. Activation of human B-cells induced by anti-IgM is associated with the induction of c-myc by greater than 20 times basal levels, maximal levels occurring at about 2 hours. This increased c-myc expression is inhibited by greater than 90% by 3 mM TMBA or 3mM HMBA. TMBA and HMBA also inhibit by greater than 90% the subsequent activation of the B-cells. These diacetylated polyamines may play a regulatory role in B-cell activation.

Spermidine labels proteins during sea urchin embryogenesis.

We have previously described the presence of a protein containing intact, covalently bound spermidine during very early embryogenesis of the sea urchin (Strongylocentrotus purpuratus). Proteins containing other polyamine metabolites also appear as embryogenesis proceeds. These proteins which contain label derived from exogenous radioactive spermidine show a characteristic pattern which changes during the course of embryonic development. We document for the first time that hypusine, the polyamine metabolite which is a characteristic component of the eukaryotic protein translation initiation factor eIF-4D, is present in more than one species of macromolecule. In addition, N1-acetylspermidine has also been identified as a significant intracellular metabolite of spermidine during embryogenesis.

Polyamines and their derivatives as modulators in growth and differentiation.

The polyamines and their derivatives are essential for life in eukaryotic and most prokaryotic cells, but their exact role in preserving cell function is not clear. These polyamines provide endogenous cations and thus participate in regulation of the intracellular pH; in addition, polyamine derivatives modulate cell growth and differentiation. The naturally occurring monoacetyl derivatives can induce increased activity of ornithine decarboxylase, the first enzyme in polyamine synthesis, and thus produce positive feedback to their production. The diacetyl derivatives of putrescine and of the synthetic analogue, 1,6-diaminohexane, induce differentiation and inhibit growth in many types of cells in vitro. In addition, they inhibit the proliferative and secretory response of normal B lymphocytes to B-cell mitogens and reduce production of antibodies in vitro. They also inhibit the proliferation of chronic lymphocytic leukemia cells (a B-lymphocyte leukemia). The parent polyamines are post-translational modifiers of proteins, and hypusine, a derivative of spermidine, is a covalently bound constituent of the eukaryotic protein synthetic initiation factor, eIF-4D. Although these various actions do not at present fall into a coherent pattern, they clearly indicate that polyamines and their derivatives play an important part in modulating cell proliferation and differentiation.

Polyamine metabolism in chronic lymphocytic leukemia.

We have previously shown that polyamines have profound effects on lymphocyte proliferation and function. We now report that the proliferative response of cultured lymphocytes from patients with chronic lymphocytic leukemia is abrogated by the addition of diacetyldiaminohexane (HMBA), the 6-carbon analogue of diacetylputrescine. In addition, this study demonstrates the post-translational modification of proteins by polyamines in CLL-lymphocytes, a decrease in the uptake of exogenous spermidine by CLL-lymphocytes which have been exposed to HMBA, and the significant conversion of spermidine to N1-acetylspermidine in CLL-lymphocytes.

Polyamines in murine splenic lymphocytes.

Diacetyldiamines cause compromised B-lymphocyte function, as evidenced by our previous demonstration of inhibition of mitogen activation and decreased secretion of immunoglobulin in murine spleen cultures. In this study, we report that putrescine and spermidine are differentially metabolized by the cell. Diacetyldiamine, which is also taken up by these cells and metabolized, causes a striking decrease in cell uptake of exogenous putrescine and spermidine. We also report for the first time that several distinct macromolecules containing radioactive polyamines may be resolved, and that hypusine is present in more than one species of macromolecule.

Binding of spermidine to a unique protein in thin-layer tobacco tissue culture.

The mechanism by which spermidine induces the appearances of floral buds in thin-layer tobacco (Nicotiana tabacum) tissue culture was studied by following the fate of the radioactive compound. [3H]Spermidine was taken up rapidly by the tissue, and after a brief lag, a portion was bound to trichloroacetic acid precipitable macromolecules. Such binding increased to a maximum on day 4 of culture, coinciding with the onset of bud differentiation, and declined thereafter until shortly before flowering. About 82% of the label in the trichloroacetic acid precipitate remained as spermidine, 14% was metabolized to putrescine, 3% to spermine, and 1% to gamma-aminobutyric acid. Spermidine was covalently bound to a protein with a molecular size of about 18 kilodaltons. Hydrolysis of this protein and analysis of the labeled entities revealed 81% spermidine, 16% putrescine, and 3% spermine. This post-translational modification of a unique protein by attachment of spermidine may be causally connected to the appearance of flower buds in thin-layer tobacco cultures.

Effects of diacetyl diamines on in vitro activation and proliferation of human B lymphocytes.

N,N'-Diacetylputrescine (tetramethylenebisacetamide [TMBA]) and its six carbon analog, hexamethylenebisacetamide (HMBA), inhibited the proliferative response of human B lymphocytes to anti-mu and formalinized Cowan I strain Staphylococcal aureus (SAC) stimulation. In contrast, B cell growth factor-stimulated proliferation of human B cells was minimally inhibited by TMBA or HMBA. The antiproliferative effect of these diamine derivatives was specific for anti-mu (or SAC) activation of normal B cells, because the proliferation of PHA-stimulated human T cells and transformed human B cells was not affected by the presence of TMBA or HMBA. The inhibitory effect of diacetyl diamines on anti-mu (or SAC)-induced B cell activation was dose dependent and persisted after removal of the diamine derivatives from the culture media. These studies show that diacetylated derivatives of polyamines modulate human B cell activation in vitro by specific abrogation of anti-mu or SAC activation.

Spermidine is bound to a unique protein in early sea urchin embryos.

Spermidine is rapidly taken up and becomes bound to protein during the very early hours of sea urchin embryogenesis. During the first 6 hr after fertilization of freshly obtained sea urchin eggs (Strongylocentrotus purpuratus), which are incubated in the presence of exogenous [3H]-spermidine, up to 7% of the total cell-associated spermidine appears uniquely as spermidine bound in macromolecular form. This unique protein containing spermidine migrates as a single radioactive band in gel electrophoresis. It has a Mr of approximately equal to 30,000 and is readily distinguishable from the protein initiation factor eIF-4D, which has a Mr of 18,000, the only other identifiable protein known to date to be posttranslationally modified by polyamines.

Polyamine metabolism in differentiating Friend erythroleukemia cells.

Changes occur in polyamine metabolism which are associated with differentiation of Friend erythroleukemia cells in culture. The intracellular distribution of the polyamines changes, the absolute level of each of the major polyamine falls, and the uptake and metabolism of exogenous putrescine is altered during the course of differentiation. We have examined the labeling of putrescine metabolites after the administration of radioactive putrescine, either as a single dose added at the onset of induction, or as a pulse label added at various times during the course of differentiation. In these experiments we measured both acid-soluble and acid-precipitable radioactivity. The conversion of putrescine to gamma-aminobutyric acid is enhanced in the cell induced to differentiate, and the acid-precipitable fraction rises as the percentage of total cell-associated radioactivity in the induced cell. We have identified hypusine [N-epsilon-(4-amino-2-hydroxybutyl)lysine] as a major radioactive component of a single radioactive Mr 18,000 protein band in both the noninduced and the induced cells.

Identification of a glycosidase activity with apparent specificity for 2-deoxy-D-glucose in glycosidic linkage.

2-Deoxy-D-glucose (dGlc) is a carbohydrate with significant activity as an inhibitor of glucose metabolism and as a precursor in the synthesis of glycosylated macromolecules; several of the enzymes associated with its metabolism remain uncharacterized. In the present report, the partial purification and some of the properties of a mammalian enzyme that appears to be relatively specific for the hydrolysis of dGlc bound in glycosidic linkage is described. The physiological function of this enzymatic activity is unknown. In addition, dGlc has been shown to be taken up by HTC cells in culture and incorporated into macromolecular bound form, both as dGlc and as 2-deoxygalactose which is formed from dGlc.

Acetylated diamines inhibit endotoxin-induced lymphocyte activation.

Fully acetylated diamines hexamethylenebisacetamide (HMBA) and diacetylputrescine (tetramethylenebisacetamide, TMBA) cause inhibition of murine B lymphocyte activation in the absence of ruminant serum. Mitogenesis induced by LPS, LAP, and 8-BrcGMP was profoundly inhibited in the presence of 3 mM HMBA and TMBA. PHA-stimulated thymidine uptake was not affected. Higher levels of the acetylated polyamines were toxic to lymphocytes. Immunoglobulin secretion by LPS-stimulated spleen cells was inhibited in similar fashion. This inhibition requires the early addition of the polyamines and does not appear to be associated with direct inhibition of DNA synthesis. These data support the hypothesis that polyamines represent immunomodulatory agents and suggest that acetylated derivatives of polyamines may also have activity in regulating B lymphocyte activation.

GABA from putrescine is bound in macromolecular form in keratinocytes.

We report for the first time that the neurotransmitter gamma-aminobutyric acid (GABA) exists in macromolecular form in keratinocytes. GABA derived from putrescine (Pu) has been identified as a component of acid-precipitable material of cultured human keratinocytes. Confluent, stratified cultures of human foreskin keratinocytes exposed to [3H]-Pu for 4 hours took up about 14% of the radioactivity from the medium and 1% of the total cell-associated radioactivity was precipitable by trichloroacetic acid (TCA). Both attached and shed cells were examined by HPLC for Pu and its radioactive metabolites in TCA-insoluble and TCA-soluble fractions. GABA accounted for the major portion (54%) of the radioactivity derived from Pu in the TCA-precipitable material of attached keratinocytes. Pu and spermidine represented lesser amounts, 35% and 9% respectively, of the total TCA-precipitable radioactivity. In addition, a large portion of acid soluble radioactivity derived from Pu (63%) was GABA, whereas Pu and spermidine represented 29% and 6% respectively of the total TCA-soluble radioactivity. The exact origin of GABA in acid-precipitable material, as well as its form of attachment, is currently under investigation.

Affinity chromatography with specific antibody increases activity and retains antigenicity of ornithine decarboxylase.

An affinity chromatography column with antibody specific to ornithine decarboxylase (ODC) was employed successfully for the retention and subsequent partial elution of ODC with enhanced activity and retention of antigenicity. This may be a useful procedure for further attempts to produce antiserum for identification and characterizations of ODC in vitro and in vivo.

Factors modulating the activity of ornithine decarboxylase in rat HTC cells.

Dibutyryl cyclic AMP (dibu-cAMP), N1-acetylputrescine and both N1-acetylspermidine and N8-acetylspermidine regularly induce ornithine decarboxylase activity in both a time- and a concentration-dependent manner when added to HTC cells in suspension culture. N,N'-Diacetyl-1,6-diaminohexane and N-acetyl-1,3-diaminopropane, aliphatic analogues of acetylated putrescine containing the carboxamide bond but not known to occur naturally in animal tissues, are also effective inducing agents. The addition of 10(-2)M putrescine to the cell culture at the time of maximal induction of ODC in HTC cells by dibu-cAMP causes a marked decrease in the apparent half-life of ODC in less than 10 minutes. Ten-fold concentration of HTC cells at the time of their maximal induction by dibu-cAMP also results in an immediate decrease both in the specific activity of ODC and in its apparent half-life. When the population density of induced HTC cells is increased the relative effect of exposure of cells to increasing levels of putrescine is lessened. Some of the implications of these observations are discussed.