Isolation and Characterization of a Murine P388 Leukemia Line Resistant to Thiarabine

A murine P388 leukemia line fully resistant to thiarabine was obtained after five courses of intraperitoneal treatment (daily for nine consecutive days). The subline was sensitive as was the parental P388/0 line to 5-fluorouracil, gemcitabine, cyclophosphamide, cisplatin, melphalan, BCNU, mitomycin C, doxorubicin, mitoxantrone, etoposide, irinotecan, vincristine, and paclitaxel, but was cross resistant (at least marginally) to three antimetabolites: palmO-ara-C, fludarabine phosphate, and methotrexate. The deoxycytidine kinase activity in the subline was comparable to that for P388/0, whereas the dCMP deaminase activity was 43% of that for P388/0. No deoxycytidine deaminase activity was detected in either of the leukemias. There appeared to be little, if any, difference in the metabolism of deoxycytidine, cytidine, or thiarabine in the two leukemias.     

Effects of cis-unsaturated fatty acids on doxorubicin sensitivity in P388/DOX resistant and P388 parental cell lines

It has been reported that several cis-unsaturated fatty acids (c-UFAs) could increase doxorubicin (DOX) accumulation in cancer cells and hence elevate its cytotoxicity. However, some researchers showed that c-UFA pretreatment did not affect its cytotoxicity in special cell lines. It is possible that the different results occurred due to different cellular characteristics. We hypothesized that c-UFA treatment might modulate the activities of some antioxidant enzymes to affect the resistance of cells to DOX. In the present study, we examined how c-UFA pretreatment affected DOX cytotoxicity on mouse leukemia cell line, P388, and its resistant subline, P388/DOX, which we found to have significantly higher glutathione peroxidase (GPx) activity as well as P-glycoprotein (p-gp) overexpression. We chose two c-UFAs, gamma-linolenic acid (GLA) (18:3n-6) and docosahexaenoic acid (DHA) (22:6n-3). Cytotoxicity was measured by MTT (3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and trypan blue exclusion assays. DOX accumulation and p-gp expression were measured by flow cytometry. The activities of catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and GPx were determined for both cell lines with and without treatment with GLA or DHA. Significant DOX accumulation occurred in both cell lines with GLA or DHA pretreatment, but without any change in p-gp expression in either cell line. Sensitivity to DOX cytotoxicity was improved by GLA or DHA pretreatment in P388/DOX in which only SOD activity was significantly increased, but not in the parental cell line P388 in which both SOD and CAT were significantly increased by the pretreatment. However, combined pretreatment of GLA or DHA with antioxidants, pyrrolidinedithiocarbamate (PDTC) or Vitamin C, could sensitize not only P388/DOX but also P388 cells to DOX. We conclude that the effects of c-UFA pretreatment on the sensitivity of cancer cells to DOX not only depend on the change in drug accumulation but also the change in the levels of antioxidant enzyme activities, and suggest that combined administration of c-UFAs, antioxidants, and DOX may be more effective in treating leukemia.     

In vitro evaluation of the chemosensitivity of an experimental murine leukemia rendered resistant in vivo to adriamycin

The results obtained using a rapid assay for in vitro chemosensitivity detection of leukemias are presented. The assay, performed according to the technique already described, involves in vitro incubation of a tumor cell suspension with various concentrations of antitumor drugs for 1 h and evaluation of drug-induced cell damage by addition to the cultured cells of 125I-deoxyuridine 48 h after pharmacological treatment. Results are expressed as percent inhibition of the isotope incorporation with respect to untreated controls. Preliminary results demonstrated that this assay is able to evidence differential chemosensitivity exhibited in vivo by murine leukemias. The present study reports the results obtained using comparatively P388 and P388/ADR, a subline of P388 murine leukemia with acquired resistance to Adriamycin in vivo. We found that P388/ADR exhibited resistance to ADR and DNR at all the concentrations tested, whereas P388 was highly sensitive. Cross-resistance of P388/ADR was also found to some structurally dissimilar agents, i.e. VCR and Act-D. These in vitro results correlate well with much data in the literature concerning the characteristics of resistance and cross-resistance exhibited in vivo by P388/ADR. These results suggest the possibility of using a similar in vitro assay for predicting the in vivo drug resistance of human leukemias.     

Specific selection of deoxycytidine kinase mutants with tritiated deoxyadenosine

We have shown previously that a low concentration of tritiated deoxyadenosine, i.e., 1 µCi/ml, selectively kills wild-type S49 murine lymphoma cells. Mutant cells resistant to [³H]deoxyadenosine lacked adenosine kinase completely but retained a significant level of deoxyadenosine phosphorylating activity. To study further the specificity of [³H]deoxyadenosine selection, lymphoma cell clones resistant to 15 µCi/ml [³H]deoxyadenosine have been derived. The resistant line, S49-dA15, is also resistant to high levels of nonradioactive deoxyadenosine and to deoxyguanosine but remains sensitive to thymidine. The thymidine inhibition of the growth of the mutant, in contrast to that of the wild-type cells, cannot be prevented by deoxycytidine. The mutant line lacks deoxycytidine kinase that also phosphorylates deoxyadenosine. In addition, the mutant cells excrete a large amount of deoxycytidine into culture medium, consistent with a failure of salvage of the nucleoside in the absence of an appropriate kinase, i.e., deoxycytidine kinase. In contrast, a deoxycytidine kinase-deficient cell line that was selected with arabinosylcytosine does not excrete deoxycytidine and contains high deoxycytidine deaminase activity. [³H]Deoxyadenosine can be used as a selective agent for specific selection of deoxycytidine kinase-negative mutants.     

Estrogen-bridged purines: A new series of anti-tumor agents which alter cell membrane properties

An estrogen-bridged adenine derivative was equitoxic to both the P388 murine leukemia and an adriamycin-resistant subline, $\text{P388}\text{ADR}$. The drug rapidly altered several P388 and $\text{P388}\text{ADR}$ membrane properties resulting in impaired nucleoside transport and increased membrane hydrophobicity. Resistance to anthracyclines in $\text{P388}\text{ADR}$ is associated with an operational barrier to drug retention which was reversed by exposure to the estrogen-bridged adenine derivative. These results suggest further exploration of the estrogen-bridged purines as chemotherapeutic agents.     

Isolation of highly multidrug-resistant P388 cells from drug-sensitive P388/S cells by flow cytometric cell sorting

To investigate the spontaneous frequency of occurrence of stable multidrug-resistant cells in a population of drug-sensitive cells, we exposed drug sensitive P388/S cells to daunorubicin (dnr) for 1 h, then used fluorescence-activated cell sorting based on intracellular dnr fluorescence to isolate cells within P388/S having different intracellular content of drug. One of the sort windows chosen (low dnr content sort window) isolated only P388/S cells with intracellular drug content equal to or less than that of the known multidrug-resistant subline P388/adr. This sort window constituted approximately 3% of P388/S cells with lowest dnr content. By such a procedure we were able, on one of seven attempts, to isolate and cultivate stable, highly multidrug-resistant cells (comparable to that of P388/adr) from the P388/S cells obtained from the low dnr-content sort window. Net growth of cells in culture was observed 15-20 days after sorting, indicating that of the P388/S cells collected from the low dnr-content sort window, very few were actually highly drug-resistant. On no occasion could resistant cells be cultivated from cells sorted from P388/S with higher dnr content, as would be expected if mutation to a multidrug-resistant phenotype had occurred as a result of exposure to drug. The resistant cells isolated from P388/S by sorting (called P388/LoSort) displayed low intracellular accumulation of dnr that was enhanced by verapamil, were cross-resistant to vincristine and actinomycin-D, and distinct from P388/S, possessed a 150- to 160-kD membrane species identified by Vinca alkaloid photoaffinity labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

A human-mouse somatic hybrid line selected for human deoxycytidine deaminase.

A new selective medium has been developed for cells containing the enzyme deoxycytidine deaminase. This medium contains hypoxanthine, aminopterin, and 5-methyldeoxycytidine (HAM medium). To survive in the presence of the aminopterin, the cells must utilize deoxycytidine deaminase to convert the 5-methyldeoxycytidine to thymidine. The cells must also have thymidine kinase and hypoxanthine phosphoribosyltransferase. A mouse cell line deficient in deoxycytidine deaminase has been isolated from a deoxycytidine kinase-deficient line, using 5-bromodeoxycytidine as the selective agent. A hybrid line between this double mutant and a human diploid fibroblast was isolated in HAM medium. The hybrid line contains the chromosomes expected of a human-mouse hybrid. The deoxycytidine deaminase isozyme patterns on cellogel show that the human-mouse hybrid cell line produces an enzyme with an electrophoretic mobility intermediate between that of the human and that of the mouse.     

Molecular basis of 2′,3′-dideoxycytidine-induced drug resistance in human cells

Human monoblastoid cells (U937) grown in the presence of therapeutically relevant dideoxycytidine concentrations (0.1 M) become resistant to the drug thanks to an altered deoxycytidine kinase. In this paper we show that deoxycytidine kinase mRNA is significally reduced in drug-resistant U937 cells (U937-R) although the deoxycytidine kinase promoter is normal. A number of nucleotide deletions, insertions and substitutions was found in the coding region of deoxycytidine kinase gene. Several identified mutations result in truncated forms of the enzyme or in the introduction of stop codons: in one case a complete lack of exon 4 was found. Thus, the deoxycytidine kinase gene accumulates mutations at a very high rate, as already reported for other cytidine analogues (i.e. Ara C ) suggesting that the design of new antiviral or anticancer drugs of the cytidine family should take into account the potential development of cell resistance as a critical factor in drug failure.     

Purification of deoxycytidine kinases from two P815 murine neoplasms and their separation from deoxyguanosine kinase

Deoxycytidine kinase, which catalyzes the phosphorylation of deoxycytidine and 1-βd-arabinofuranosylcytosine (Ara-C) at the 5′-position, has been extracted and extensively purified from a murine neoplasm P815, either sensitive (P815) or resistant (P815/ Ara-C) to 1-β-d-arabinofuranosylcytosine. Gel filtration and ion-exchange chromatography were used to accomplish the purification. The purified enzyme exhibited a single band upon disc electrophoresis. During the extraction procedure an enzyme catalyzing the phosphorylation of deoxyguanosine and deoxyadenosine was successfully separated for the first time from deoxycytidine kinase. The K_m values and turnover numbers with deoxycytidine as phosphate acceptor for the kinase from P815 cells sensitive to 1-β-darabinofuranosylcytosine and that from P815 cells resistant to the drug are 9.3 μm, 4.7 × 10⁶/min and 15.4 μm, 8.0 × 10⁴/min, respectively.     

Modulation of mitomycin C resistance by glutathione transferase inhibitor ethacrynic acid.

This study was undertaken to elucidate the mechanism(s) of cross-resistance (4.9-fold) to mitomycin C (MMC) in a multi-drug-resistant cell line, P388/R-84. Intracellular accumulation of MMC by sensitive (P388/S) and P388/R-84 cells was comparable. Despite a 32% reduction in NADPH cytochrome P-450 reductase activity (responsible for MMC activation) in P388/R-84 cells, the rate of MMC bio-reduction by sensitive and resistant cells was similar. These results suggested that MMC resistance in P388/R-84 cell line must depend on factors other than impaired drug accumulation or bio-activation. Recent studies suggest that glutathione transferase (GST) dependent drug detoxification also contributes to cellular resistance of a variety of alkylating agents. Even though overexpression of GST has been noted in some MMC resistant tumor cells, it is not known if its level affects sensitivity to MMC. We have, therefore, determined the effect of ethacrynic acid (an inhibitor of GST activity) treatment on MMC cytotoxicity in P388/R-84 cells, which have about 2-fold higher GST activity than P388/S cells. The IC50 value for the inhibition of GST activity in vitro by ethacrynic acid (EA) was 16.5 microM (5 micrograms/ml). A depletion in intracellular GSH was also observed by treating P388/R-84 cells with EA alone or in combination with MMC. A non-toxic concentration of EA (1 microgram/ml; 3.3 microM) increased MMC cytotoxicity by 36% in P388/R-84 cells. MMC cytotoxicity was increased 2-fold by EA treatment in glutathione (GSH)-depleted P388/R-84 cells. These results suggest that GST mediated drug inactivation may represent another important mechanism of MMC resistance.     

Induction of acetyl-LDL receptor activity by phorbol ester in human monocyte cell line THP-1

Human monocytic cell line THP-1 incubated with as little as 10 ng/ml of phorbol myristate acetate bound and metabolized 1-2 micrograms of Ac-LDL over a 5-h period. In the absence of phorbol treatment, no specific metabolism of Ac-LDL occurred. Optimal levels of receptor were reached after 72 h of exposure. Induction of receptor was dependent on protein and RNA synthesis and was partially reversed upon removal of the phorbol. Induction of receptor required activation of the protein kinase C pathway. Metabolism of Ac-LDL by THP-1 cells at 37 degrees C was saturated at 25 micrograms/ml. Binding at 4 degrees C was saturable with an average Kd of 8.0 x 10(-9) M. Cell population studies by fluorescent activated cell sorting indicated that approximately 87% of the THP-1 population was expressing scavenger receptor activity 96 h after phorbol treatment as compared to 99% for murine macrophage cell line P388D1. Uptake of Ac-LDL by THP-1 resulted in an 11-fold increase in the rate of cholesterol esterification which was saturable at 50 micrograms/ml. Incubation of cells for 48 h with 50 micrograms/ml of Ac-LDL resulted in a 60% increase in free cholesterol and a 10-fold increase in the cholesteryl ester content of the cells. Lipid accumulation in THP-1 cells after Ac-LDL uptake was readily visible by Oil Red-O staining. Solubilization of THP-1 cells, before and after phorbol treatment, followed by ligand blotting with Ac-LDL detected the presence of a 250-kDa protein only in cells treated with phorbol. The protein comigrated with the scavenger receptor derived from mouse macrophage cell line P388D1.     

Cell surface glycoproteins involved in the stimulation of interleukin 1-dependent interleukin 2 production by a subline of EL4 thymoma cells. I. Functional characterization by monoclonal antibodies

Three rat monoclonal antibodies (MAb) capable of stimulating interleukin 2 (IL 2) production by a variant subline of EL4 thymoma cells (EL4-6.1) have been produced. The stimulatory capacity of these MAb (designated RL73, RL119, and RL388) was originally found to be dependent on the presence of irradiated peritoneal exudate cells; however, this requirement could be replaced by the cellfree supernatant of the "macrophage-like" cell line P388D1 or by biochemically purified human interleukin 1 (IL 1). A number of other rat MAb directed against cell surface structures did not stimulate IL 1-dependent IL 2 production by EL4-6.1 cells; however, certain MAb directed against Thy-1 as well as the lectin phytohemagglutin did have this capacity. Furthermore, the stimulatory activity of MAb RL73, RL119, and RL388 appeared to be restricted to the EL4-6.1 variant line, because neither the parental EL4 line from which it was derived nor a series of ovalbumin-specific T-T hybrids responded to these MAb. The cell surface antigens recognized by MAb RL73, RL119, and RL388 were present on a wide variety of T cell lines and T-T hybrids, as well as on lines of B cell, macrophage, and fibroblast origin. Interestingly, the MAb reacted with the majority (approximately 85%) of thymocytes but not (or only to a very small extent) with resting T lymphocytes. After stimulation by concanavalin A, however, the three MAb reacted strongly with activated T lymphoblasts. The latter data suggest that MAb RL73, RL119, and RL388 may react with cell surface structures that are normally expressed as a consequence of lymphocyte activation.     

Protein kinase activity associated with Fc gamma 2a receptor of a murine macrophage like cell line, P388D1

The properties of protein kinase activity associated with Fc receptor specific for IgG2a (Fc gamma 2aR) of a murine macrophage like cell line, P388D1, were investigated. IgG2a-binding protein isolated from the detergent lysate of P388D1 cells by affinity chromatography on IgG-Sepharose was found to contain four distinct proteins of Mr 50,000, 43,000, 37,000, and 17,000, which could be autophosphorylated upon incubation with [gamma-32P]ATP. The autophosphorylation of Fc gamma 2a receptor complex ceased when exogenous phosphate acceptors (casein or histone) were added in the reaction mixture. Casein was found to be a much better phosphate acceptor than histone in this system, as casein incorporated about 32-fold more 32P than histone did. Phosphorylation of casein catalyzed by Fc gamma 2a receptor complex was dependent on casein concentration (maximum phosphate incorporation being at 0.5 mg/mL), increased with time or temperature, was dependent on the concentration of ATP and Mg2+, and was maximum at pH near 8. Casein phosphorylation was significantly inhibited by a high concentration of Mn2+ (greater than 25 mM) or KCl (greater than 100 mM) or by a small amount of heparin (greater than 10 units/mL) and was enhanced about 2-fold by protamine. Casein kinase activity associated with Fc gamma 2a receptor used ATP as substrate with an apparent Km of 2 microM as well as GTP with an apparent Km of 10 microM. Prior heating (60 degrees C for 15 min) or treatment with protease (trypsin or Pronase) of Fc gamma 2a receptor complex almost totally abolished casein kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase

The kinetic properties of 50,000-fold purified cultured human T lymphoblast (MOLT-4) deoxycytidine kinase were examined. The reaction velocity had an absolute requirement for magnesium. Maximal activity was observed at pH 6.5-7.0 with Mg:ATP for 1:1. High concentrations of free Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both deoxycytidine and MgATP2-. dCMP was a competitive inhibitor with respect to deoxycytidine and ATP. ADP was a competitive inhibitor with respect to ATP and a mixed inhibitor with respect to deoxycytidine. dCTP, an important end product, is a very potent inhibitor and was a competitive inhibitor with respect to deoxycytidine and a non-competitive inhibitor with respect to ATP. TTP reversed dCTP inhibition. The data suggest that (a) MgATP2- is the true substrate of deoxycytidine kinase; (b) the kinetic mechanism of deoxycytidine kinase is consistent with rapid equilibrium random Bi Bi; (c) deoxycytidine kinase may be regulated by its product ADP and its end product dCTP as well as the availability of deoxycytidine. While many different nucleotides potently inhibit deoxycytidine kinase, their low intracellular concentrations make their regulatory role less important.     

Low level of mitochondrial deoxyguanosine kinase is the dominant factor in acquired resistance to 9-beta-D-arabinofuranosylguanine cytotoxicity

9-beta-D-arabinofuranosylguanine (Ara-G) is an important and relatively new guanosiue analog with activity in patients with T-cell malignancies. The biochemical and molecular events leading to resistance to Ara-G are not fully understood. Therefore we generated two Ara-G-resistant human MOLT-4 leukemic cell lines with different levels of resistance. The mitochondrial enzyme deoxyguanosine kinase (dGK) and the nuclear/cytosol enzyme deoxycytidine kinase (dCK) are key enzymes in the activation of Ara-G. Decreased levels of dGK protein and mRNA were found in both resistant cell sublines. The activity of dCK was decreased in the subline with higher resistance to Ara-G and these cells were highly cross-resistant to other nucleosides activated by dCK. Increased activity of the mitochondrial enzyme thymidine kinase 2 was observed in both resistant sublines and this could be related to the dGK deficiency. In search for other resistance mechanisms it was found that the resistant cells overexpress the mdr1 gene, while no changes were detected in the levels of multidrug resistance-associated protein 1 through 6, lung resistance-associated protein or topoisomerase IIalpha or IIbeta. Taken together, our findings demonstrate that multiple mechanisms are involved in the acquired resistance to Ara-G. However, low expression of dGK is the most apparent alteration in both resistant cell lines. Partial deficiency of dCK was found in the subline cells with higher resistance to Ara-G. Furthermore, Ara-G may select for high expression of the multidrug resistance (mdr1) which could be a specific resistance mechanism but more likely part of an overall cellular stress response.     

Adherent Ia+ murine cell lines with characteristics of dendritic cells. II. Characteristics of I region-restricted antigen presentation

The ability of an adherent Ia+, interleukin 1+ (IL-1) tumor cell line (P388AD) to present turkey gamma-globulin (TGG) to primed T lymphocytes was demonstrated and compared with normal antigen-presenting cells (APC) found in mouse spleen. P388AD tumor cells presented TGG to long-term cultures of TGG-reactive T cells (LTTC) and to lymph node-derived T cells which were enriched on nylon wool columns and subsequently depleted of endogenous antigen-presenting cells with anti-Ia antisera and complement. MHC-restricted antigen presentation by P388AD was observed when long-term cultures of TGG-reactive T cells were used as the responding T-cell population. Furthermore, antisera directed against I-region determinants expressed on the P388AD tumor cells inhibited TGG-specific T-cell proliferation in a dose-related fashion, suggesting a functional role for the tumor cell-associated Ia molecules. The kinetics of antigen presentation to LTTC by P388AD were similar to the kinetics observed for splenic APC, although the magnitude of the proliferative response to LTTC to TGG was generally lower when antigen (Ag) was presented by the tumor cells compared to splenic antigen-presenting cells (APC). However, the magnitude of T-cell proliferation of immune lymph node (LN) T cells was comparable when Ag was presented on tumor cells or splenic APC. Several experiments suggested that Ag uptake and/or processing may be less effective in P388AD tumor cells as compared to normal splenic APC. A nonadherent Ia+, IL-1- tumor cell line (P388NA), which was isolated from the same parental tumor as P388AD, was also tested for the ability to present Ag to primed T lymphocytes and Ag-reactive LTTC. In contrast, to P388AD, the nonadherent tumor cell failed to present TGG under identical culture conditions even though Ia molecules were expressed on the tumor cells and Ag uptake had occurred. However, the defect in Ag presentation by P388NA could be corrected if an exogenous source of purified interleukin 1 was supplied to the cultures. A unique opportunity thus exists with both the P388AD and P388NA tumor cell lines to decipher some of the molecular interactions leading to T-cell proliferation during antigen presentation.     

Reversion in thymidine kinase deficient variants of mouse lymphoma P388

The ability of 5 independently isolated thymidine kinase-deficient clones of mouse lymphoma P388 to revert has been examined. We were unable to detect spontaneous revertants in any of the 5 clones. Treatment with the hypomethylating agent 5-azacytidine induced reversion in 4 of the clones, but the frequency of revertants was very low (less than 10(-6). The response was not dose-dependent. The mutagen EMS was capable of inducing reversion in 3 of the clones with a variable level of response. The activity of thymidine kinase in 16 revertants was determined. In half of these the level of enzyme activity was considerably greater than the original P388 cell line. The high frequency loss of thymidine kinase that occurs in these cells may represent a stable inactivation of gene activity rather than an alteration in the DNA base sequence.     

The effect of finoptin on doxorubicin accumulation in leukemia P-388 cells with induced resistance to the combination of finoptin and doxorubicin

Using hybrid mice BDF1 doxorubicin (Dx) accumulation has been determined in leukemia P388 cells (P388/0), P388 cells with induced resistance to Dx (P388/Dx) and P388 cells with induced resistance to the finoptin (Fp) + Dx combination (P388/Fp + Dx). It has been shown that Fp doesn't affect Dx accumulation in or elimination from leukemia cells P388/0 or P388/Fp + Dx. The resistance of P388/Fp + Dx cells to the Fp + Dx combination develops during 6 passages. It can be concluded that Fp application doesn't abolish the problem of tumor cells' resistance to cytostatics.     

Induction of functional Fc receptors in P388 leukemia cells : Requirement for multiple differentiation signals

The development of functional Fc receptors (FcR) during induced differentiation with the tumor promoter, phorbol myristate acetate (PMA), was studied in the murine tumor cell line, P388. PMA induced the appearance of FcR on the membranes of P388 cells as indicated by the binding of IgG-coated sheep red blood cells (IgG-SRBC). Concentrations of PMA as low as 1 ng/ml were sufficient to induce the expression of FcR as well as to inhibit cellular division and to induce adherence in the P388 tumor cell line; however, optimal FcR induction occurred at PMA concentrations of 10-100 ng/ml. Immunofluorescent analysis with heat-aggregated myeloma proteins indicated that PMA induced FcR which were capable of binding IgG2a and IgG2b immunoglobulins, but not IgG1. Adherence to a substratum was determined to be a second required signal for expression of FcR, since PMA induction of P388 tumor cells in teflon dishes failed to fully develop FcR and adherence of P388 cells to poly-L-lysine-coated culture dishes in the absence of PMA was insufficient for FcR expression. FcR which appeared after PMA induction were non-functional in the sense that membrane-bound IgG-SRBC were not ingested to any significant extent by the tumor cells. However, if FcR induction occurred in the presence conA-induced rat spleen cell culture supernatants, phagocytosis of membrane-bound erythrocytes occurred. These findings suggest that for the expression of FcR which are capable of particle internalization, at least three identifiable membrane-transmitted signals are required during differentiation.     

Deoxyadenosine/deoxycytidine kinase from Bacillus subtilis. Purification, characterization, and physiological function

Three different deoxyribonucleoside kinases with specificities toward thymidine, deoxyguanosine, and deoxyadenosine/deoxycytidine, respectively, are identified in Bacillus subtilis. The deoxyadenosin/deoxycytidine kinase is purified 950-fold employing blue Sepharose CL-6B column chromatography. The two deoxyribonucleoside kinase activities copurify and are present in the same band after polyacrylamide gel electrophoresis. The molecular weight is determined by gel filtration to be 47,000. Cytidine, adenosine, arabinosylcytosine, and arabinosyladenine are substrates for the enzyme. The activities toward these substrates are less than 20% of the activities obtained with deoxyadenosin and deoxycytidine. The deoxycytidine and deoxyadenosine saturation curves are hyperbolic with Km values for both nucleosides around 5 microM. The maximal velocities for the two deoxyribonucleosides are nearly identical with GTP as phosphate donor. GTP is the best donor showing hyperbolic saturation curves and Km values around 150 microM depending on the deoxyribonucleoside concentration. dATP and dCTP are inhibitors when GTP is the phosphate donor. They may both act as phosphate donors themselves. A divalent metal ion is required, Mg2+ giving the highest activity. A spontaneous mutant, selected as resistant to 5-fluorodeoxycytidine, lacks both deoxycytidine and deoxyadenosine kinase activity, while it retains normal activities toward deoxyguanosine, deoxyuridine, and thymidine.