1 Max-Planck-Institut für Molekulare Physiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany
2 Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires, Université Pierre et Marie Curie, Hôpital de la Pitié-Salpêtrière, CNRS ERS 107 CERVI, 83 boulevard de l'Hôpital, F-75651 Paris Cedex 13, France
3 Max-Planck-Institut für Biophysikalische Chemie, Abteilung Molekulare Genetik, Am Fassberg 11, D-37077 Göttingen, Germany
Correspondence
Manfred Konrad
mkonrad{at}gwdg.de
Birgitta M. Wöhrl
birgitta.woehrl{at}uni-bayreuth.de
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ABSTRACT |
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Present address: Universität Bayreuth, Lehrstuhl Biopolymere, Universitätsstr. 30, D-95447 Bayreuth, Germany.
Present address: Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, PO Box 19024, Clinical Division D2-100, Seattle, WA 98109, USA.
These authors contributed equally to this work.
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INTRODUCTION |
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AZT is an important antiviral prodrug used in the treatment of human immunodeficiency virus (HIV) infections. The antiviral activity of AZT is due to AZT triphosphate (AZTTP), which acts as a chain terminator during reverse transcription of the viral RNA genome. After uptake of the nucleoside analogue AZT into the cell (Zimmerman et al., 1987), cellular enzymes phosphorylate AZT to its triphosphate form in a stepwise manner (Furman et al., 1986
). The first enzyme in this pathway is cellular thymidine kinase (TK), which uses ATP to catalyse the phosphorylation of AZT to its 5'-monophosphate (AZTMP). The second enzyme, thymidylate kinase (TMPK), converts AZTMP to AZT diphosphate (AZTDP), a substrate for nucleoside diphosphate kinase, which is assumed to catalyse the AZTDP to AZTTP phosphorylation. The prodrug AZT is readily converted into AZTMP, which then accumulates in the cell, resulting in low intracellular concentrations of AZTDP and AZTTP (Furman et al., 1986
). The bottleneck in AZT activation was shown to be the addition of the second phosphoryl group catalysed by TMPK (Lavie et al., 1997a
).
Our recent structural and biochemical studies of TMPKs from various sources demonstrated that in contrast to yeast and human TMPK, the TMPK from Escherichia coli can phosphorylate AZTMP very efficiently (Brundiers et al., 1999). The primary and tertiary structures of the yeast and human TMPKs are quite similar whereas the E. coli TMPK exhibits structural differences that appear to be important for its efficient phosphorylation of AZTMP (Lavie et al., 1997a
, b
, 1998a
, b
; Ostermann et al., 2000a
, b
). Kinetic analyses of the enzymes revealed that the catalytic rate constant, kcat, for AZTMP phosphorylation is about 35-fold higher with E. coli TMPK as compared with yeast TMPK, and about 500-fold higher as compared with human TMPK. A summary of these data is shown in Table 1
.
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METHODS |
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The retroviral integration vector used is a derivative of the Moloney murine leukaemia virus (Mo-MuLV) containing the encapsidation signal , the gene encoding the human surface protein Thy-1 (used for flow cytometry detection) and the internal ribosome entry site (IRES) from the encephalomyocarditis virus allowing for the expression of a second recombinant gene from the same transcript. These regions are flanked by the viral Mo-MuLV long terminal repeats (LTRs). The wt or mutant human TMPKs were PCR-amplified to introduce the proper restriction sites and cloned behind the IRES sequence.
Cell lines and cell culture.
The P4CCR5 cell line, kindly supplied by P. Charneau (Institut Pasteur, Paris, France), is a HeLa-CD4 cell derivative that can be infected with HIV (Charneau et al., 1992). It carries the bacterial lacZ gene under the control of the HIV-1 LTR sequence. P4CCR5 cells also express the CCR5 co-receptor for infection by macrophagic HIV-1 strains and the CXCR4 co-receptor for infection by lymphotropic HIV-1 strains. The expression of
-galactosidase was used as a marker for HIV infection and was detected 48 h post-infection. The promonocytic leukaemic human cell line U-937 was obtained from the ATCC (Manassas, VA, USA); it expresses the CD4+ receptor but not the Thy-1 surface protein (CD4+ Thy-1). TE-FLY GA16 (CD4 Thy-1), supplied by F.-L. Cosset (Cosset et al., 1995
), is a Mo-MuLV based human packaging cell line that produces high titres of retrovirus pseudotyped with gibbon ape leukaemia virus envelope.
The cell lines P4CCR5 and TE-FLY GA16 were grown in DMEM (Dulbecco's modified Eagle's medium; GibcoBRL) containing 10 % fetal calf serum (FCS), 2 mM L-glutamine (GibcoBRL), 50 µg penicillin ml1, 100 µg neomycin ml1 and 50 µg streptomycin ml1 (GibcoBRL). The U-937 cell line was usually grown in suspension in RPMI 1640 medium (GibcoBRL) with the same additions as above; when co-cultured with the packaging cell line DMEM was used.
Transfection.
Cells (3x106) were seeded in a Petri-dish and transfected with a total amount of 20 µg plasmid DNA by using the calcium phosphate method (Sambrook & Russell, 2001). To obtain stable clones we used 1 µg of a plasmid conferring hygromycin resistance and 19 µg of the plasmid containing the recombinant gene. Medium was changed 24 h after transfection, and cells were put under hygromycin selection 48 h after the transfection. After 3 weeks, independent hygromycin-resistant clones were isolated and expanded individually.
Transient transfections of P4CCR5 cells were done following the same procedure, with pCi-human TMPK and pCi-EGFP (encoding enhanced green fluorescent protein) plasmids, using a molecular ratio of 1 : 1 (3x1012 molecules of each plasmid). Cells were harvested 48 h after transfection. Fluorescence was measured by flow cytometry of appropriate cell aliquots, and cells were then used to analyse thymidine and AZT phosphorylation as described below.
Transient transfections of P4CCR5 cells using the retroviral vector were done with 20 µg plasmid. Cells were harvested 48 h after transfection. The percentage of transfected cells was identified by analysis of Thy-1 expression (see below). Transfection efficiency was >80 % in all cases.
Production of stable U-937 clones expressing recombinant human TMPK.
Cells of the human packaging cell line TE-FLY GA16 were transfected with the retroviral vector containing wt or one of the mutated human TMPK genes as described above. After 60 h, the medium was removed and 5 ml U-937 cells were added to the culture at a concentration of 1x106 cells ml1 in DMEM culture medium. Protamine sulfate was added at a final concentration of 4 µg ml1. After 24 h, U-937 cells from the supernatant were centrifuged (1300 r.p.m. for 5 min at 20 °C) and resuspended in DMEM containing protamine sulfate (20 µg ml1). One-third to one-half of the cells were again co-cultured with the transfected packaging cells. This procedure was repeated for 10 days. Then the infected cells (CD4+ Thy-1+) were separated from the non-infected (CD4+ Thy-1) and packaging cells (CD4 Thy-1) by cell sorting (FACStar Plus; Becton Dickinson). Cells were stained by using a mouse IgG1 anti-human Thy-1 antibody (Génopoïétic) followed by a secondary goat anti-mouse IgG1 FITC-conjugated mAb (Caltag) and an anti-human CD4 mAb (CD4-Cy-Chrome). Excitation was done at 488 nm with 5-W argon laser (Coherent Palo Alto) operating at 150 mW. Emission of FITC and Cy-Chrome were measured at 518 nm and 682 nm, respectively. Cells were sorted at the rate of 3000 events s1, with the abort rate being about 10 % of the events. CD4+ Thy-1+ expressing cells were collected in 50 % FCS and afterwards proliferated in RPMI 1640 culture medium. After several weeks in culture, the cells were repurified, and after further growth the polyclonal population was used for analysis.
Analysis of Thy-1 expression by flow cytometry.
Adherent cells were trypsinized, washed with PBS (1x PBS, 100 mM NaCl, 20 mM Tris/HCl, pH 7·4), and the cell concentration was adjusted to 1x106 cells ml1 for adherent cells or to 37x106 for suspension cells. Cells were incubated for 30 min at 4 °C in 100 µl 1x PBS, BSA (1 g l1) and 0·2 g sodium azide l1 to which a murine IgG1 anti-human Thy-1 mAb was added in a 100-fold dilution. After washing, cells were incubated with the secondary mAb, FITC-labelled goat anti-mouse IgG-1 (Caltag), for 30 min at 4 °C. The number of fluorescent cells and the fluorescence intensity were determined by flow cytometry (FACSCalibur; Becton Dickinson) using Cellquest software.
Analysis of thymidine and AZT metabolism in cell lines overexpressing human TMPKs.
U-937 (5x106) or P4CCR5 cells (2x106) were cultivated for 7 h in the presence of 1 µM [methyl-3H]thymidine [5 Ci (185 GBq) mmol1; Amersham] or [methyl-3H]AZT [11·7 Ci (432·9 GBq) mmol1, Isotopchim]. At the end of the incubation time, the adherent cells were treated with trypsin. Cells were washed three times with 1x PBS. To extract the nucleotides, the cell pellets were resuspended in 1 ml ice-cold 60 % methanol, vortexed and stored at 20 °C overnight. After centrifugation for 30 min at 12500 r.p.m. (15 000 g) in an Eppendorf centrifuge, the supernatant was lyophylized in a Speed-vac and the dried pellet was resuspended in 500 µl dH20. After filtration through a 0·22 µm filter (hydrophilic Durapore PVDF low protein binding-membrane; Millipore Corporation) the suspension was stored at 80 °C.
Different forms of phosphorylated nucleotides were separated by anion-exchange chromatography of the aqueous solution by using a MonoQ HR5/5 column (Pharmacia). The bound nucleotides were eluted with ammonium phosphate buffer (25 % ammonium solution, 85 % orthophosphoric acid; pH 7·0) by using an improved gradient programme in the concentration range of 0·020·5 M phosphate (Guettari et al., 1997; Kremmer et al., 1989
). The non-phosphorylated and phosphorylated forms were analysed by counting the radioactivity at the column exit using a Radiomatic Flo-one Beta A-500 counter and were quantified by using the Flo-one/Data software (Radiomatic Flo-one/beta; Packard) (Guettari et al., 1997
).
Infection of P4CCR5 cells with HIV-1Lai-791.
The HIV-1Lai-791 strain used for infection of P4CCR5 cells was purchased from Diagnostics Pasteur. Its virus titre on P4CCR5 cells was 7·3x105 TCID50 ml1 (tissue culture 50 % infectious dose). One day before infection, about 9000 cells per well were seeded into a 96-well microtitre culture plate. On the day of infection, the cells were pre-incubated in triplicate for 5 h with different concentrations of AZT, varying from 105 to 10 µM. Infections were done in a volume of 100 µl (50 µl AZT solution plus 50 µl virus solution) in the presence of 20 µg DEAE-dextran ml1. After 24 h, 100 µl new AZT medium was added to the culture. Infections were stopped after 48 h, and -galactosidase activity of the infected cells was quantified by a chemiluminescent test (Roche Diagnostics). The
-galactosidase activity of cells infected with HIV-1Lai-791 in the absence of AZT was used as a control.
Analysis of AZT toxicity.
Non-transfected P4CCR5 cells (5x103) were cultivated in 6-well plates in the presence of different concentrations of AZT until they were weakly confluent. After 7 days in culture, cells were trypsinized, washed with 1x PBS and the viable cells were counted by using trypan blue exclusion for detection.
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RESULTS |
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To analyse the engineered TMPKs in human cells we used two cell lines (U-937 and P4CCR5) and two different expression systems: (i) a non-integrating vector containing a CMV promoter leading to constitutive expression of the recombinant gene, and (ii) a retroviral vector harbouring a double expression cassette resulting in the constitutive expression of a marker gene (encoding the Thy-1 surface protein) and, via an IRES sequence, of a second gene which in our case is human TMPK. Data shown in the figures are representative of two independent experiments. In the first step, we tested the functionality of the mutant enzymes by transient transfection of the corresponding plasmids into a human cell line.
Functionality of wt and mutant human TMPKs in P4CCR5 cells after transient transfection
Transient expression of human TMPKs via the retroviral LTR promoter.
To study the behaviour of the TMPKs in a human cell line, we constructed a retroviral vector that allows both transient transfection of human cells via transfection of the vector containing the retroviral LTR promoter, and, furthermore, infection of human cells in order to introduce the recombinant human TMPK gene stably into the host genome (see below).
The vector we constructed is based on the Mo-MuLV retrovirus. Expression of the recombinant gene was driven by the LTR promoter. The different human TMPK genes were co-expressed with a marker gene encoding the human cell surface protein Thy-1 that can easily be detected on the surface of transfected cells by flow cytometry (see Methods). Moreover, this marker protein would not cause an immune reaction in the human host, an important aspect to consider in potential therapeutic applications. Transfection efficiencies were around 80 % in all cases.
To analyse whether the recombinant human TMPK proteins were functional when expressed from this cassette, P4CCR5 cells were transiently transfected, and AZT and thymidine phosphorylation profiles were determined 7 h post-transfection. Fig. 1(a) shows typical elution profiles obtained after FPLC anion-exchange chromatography of the radioactive nucleosides and nucleotides isolated from cells grown in the presence of [3H]AZT. Levels of thymidine triphosphate (TTP) were only slightly increased in cells expressing one of the recombinant human TMPKs indicating that phosphorylation of the physiological substrate is insignificantly affected (Fig. 1b
). AZT phosphorylation, however, improved drastically when one of the mutant human TMPK enzymes was expressed (Fig. 1c
). The AZTMP proportion dropped from 90 % in the parental cells to 36 or 17 % in the presence of human TMPK F105Y or human TMPK LL, respectively. Upon expression of the wt enzyme, AZT phosphorylation also increased, though to a much lesser extent, and the AZTMP level (84 %) remained almost as high as in the parental cells (90 %).
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Cells transfected with wt or mutant human TMPK plasmids showed slightly increased thymidine phosphorylation activities, as indicated by the increased percentage of the phosphorylated species (TMP; thymidine diphosphate, TDP and TTP) in Fig. 2(a). The phosphorylation pattern of the parental cells in Fig. 2(a)
is somewhat different from that of Fig. 1(b)
. This might be due to the fact that endogenous TMPK is a cell cycle-regulated enzyme that is highly expressed only during the S-phase (Huang et al., 1994
). Since the cells are not synchronized they might have different phosphorylation patterns.
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Analysis of TMP and AZTMP phosphorylation in stable clones expressing wt or mutant human TMPKs
The central feature of a future enzyme/prodrug-based antiviral therapy is the delivery of a gene encoding the drug-activating enzyme into human cell lines and permanent integration into the host genome. To obtain more information on the AZTMP phosphorylation efficiency of the recombinant human TMPKs we decided to stably introduce the corresponding genes into the human cell lines U-937 and P4CCR5 in order to determine whether the type of cell and the vector influence the phosphorylation patterns.
Thymidine and AZT phosphorylation in the human cell line U-937 expressing recombinant human TMPK.
Retroviral constructs expressing the wt human TMPK or the F105Y mutant were used for transfection of a human packaging cell line, which in turn was co-cultured with U-937 cells. U-937 is a promonocytic leukaemic human cell line that is more similar to the cells of the immune system that are naturally infected with HIV than P4CCR5. Infection of U-937 cells with the viral vector and further purification of the infected cells by cell sorting based on Thy-1 and CD4 expression led to the isolation of a polyclonal population of cells expressing either the wt or the TMPK F105Y mutant protein. Purified cells were proliferated for several weeks and then repurified, again using Thy-1 as a selection marker.
Expression of wt or mutant TMPK did not change the thymidine phosphorylation profile (data not shown). Cells expressing wt TMPK had phosphorylation levels of AZT similar to those of the parental cells. Expression of TMPK F105Y induced a slight but significant increase of the proportions of AZTDP and AZTTP. The relative amounts of AZTDP and AZTTP were lower than in P4CCR5 cells transiently transfected with the same retroviral vector (Fig. 3a).
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Antiviral activity of AZT in a human cell line expressing a mutant human TMPK
To analyse HIV infection of cells that express recombinant human TMPK we used the P4CCR5 cell line. It harbours the bacterial lacZ gene under the control of the HIV-1 LTR and is permissive to HIV infection. The level of infection can easily be followed by determination of the -galactosidase activity of the culture.
Cytotoxic effect of AZT.
Since AZTTP can also serve as substrate for cellular polymerases, we first determined whether higher levels of intracellular AZTTP would result in increased AZT toxicity. The cytotoxic effect of AZT, which may also be due to its metabolite AZTMP, was analysed with parental cells and clones stably expressing one of the mutated human TMPKs. Determination of LD50 yielded similar values for the parental P4CCR5 cells and the clones expressing the mutated human TMPKs F105Y and LL (between 20 and 30 µM AZT), indicating no significant modification of AZT toxicity by expression of mutant TMPKs.
HIV infections of stable P4CCR5 clones.
To see whether the enhanced levels of intracellular AZTTP could be correlated with an improved antiviral activity of AZT, stable clones of P4CCR5 expressing recombinant human TMPK were infected with different dilutions of HIV-1Lai-791 virus in the presence of increasing concentrations of AZT. Two days after the infection, the -galactosidase activities of the cultures were measured. Comparison of the AZT concentrations that inhibit virus replication by 50 % (IC50), with control cells infected in the absence of AZT, shows that AZT is five to seven times more efficient in preventing HIV replication in stable clones expressing either of the two mutant human TMPKs. Whereas IC50 values for AZT lie between 0·15 and 0·22 µM in the parental cells these values are between 0·03 and 0·04 µM AZT in cells expressing the TMPK mutants (Fig. 4
).
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DISCUSSION |
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At present, we do not know at the molecular level the reasons for the less than optimal properties of intracellularly expressed human TMPK mutant enzymes that proved to be highly efficient in AZTMP to AZTDP conversion in vitro. Since human TMPK is a regulated enzyme that normally is only expressed during the S-phase of the cell cycle (Huang et al., 1994), continuous overexpression of human TMPK over a long period of time might lead to changes in cellular metabolism. Stable expression of the recombinant gene might therefore select for clones expressing human TMPK at lower levels that do not interfere strongly with the nucleotide metabolism and its regulation. It has been suggested that introduction and overexpression of a modified human TMPK in human cells cannot lead to improved AZTMP phosphorylation since AZTTP and TTP are also allosteric inhibitors of the cytosolic thymidine kinase (TK) at physiological concentrations (Balzarini et al., 1998
). Thus, increasing the intracellular AZTTP and TTP levels might have a suppressive feed-back effect on the conversion of AZT to AZTMP and, as a consequence, on the synthesis of the antivirally active AZTTP. Yet our results of low AZT levels and high levels of the phosphorylated AZT forms in the transient transfections, as well as previous results (Guettari et al., 1997
), argue against this scenario. Furthermore, because of similar Km values of the two competing substrates TMP and AZTMP, their discrimination by TMPK is determined by the ratio of the rates (vTMP versus vAZTMP) at which they are phosphorylated: vTMP/vAZTMP=(kcat/Km)TMP[TMP]/(kcat/Km)AZTMP[AZTMP]. Then, at comparable substrate concentrations, the determining factor is the catalytic rate constant, kcat, which in the case of our best mutant enzyme is higher for AZTMP than for TMP.
We have shown that even a small rise of the intracellular AZTTP level can result in a significantly increased inhibitory effect on HIV replication. This is expected considering the mechanism of HIV reverse transcriptase inhibition which implies a steep linear dependence on AZTTP concentration (Goody et al., 1991). Alternative interpretations of the increased inhibition of HIV replication may also apply in this system, notably reduced ATP-mediated AZT excision and pyrophosphorolysis (for a recent review of this issue, see Goldschmidt & Marquet, 2004
).
Our work also suggests that it could be advantageous to integrate an inducible gene expression system (Rossi & Blau, 1998) that allows for controlled production of the recombinant enzyme. Then, expression of human TMPK would only be switched on when it is actually needed, i.e during HIV infection, but not during the phase of in vitro manipulation of the cells to be genetically modified. Upon HIV infection, cells harbouring and overexpressing the recombinant huTMPK gene should be protected against the virus due to their higher intracellular level of inhibitory AZTTP. These cells, being resistant to HIV infection, will have a growth advantage over cells that do not express the recombinant gene and will repopulate the reservoir of cells destroyed by the virus. In patients, ultimately, enhanced AZTMP phosphorylation efficacy should help to suppress virus replication drastically or at least prolong the interval of emergence of drug-resistant viruses. Furthermore, expressing modified human enzymes instead of enzymes of viral, bacterial or yeast origin might be advantageous since an immune response to the introduced human protein appears less likely. Though at the present time the application of gene therapy to the treatment of AIDS may not necessarily be the most convincing example of disease management by gene/enzyme transfer, we believe that our studies on catalytically optimized human thymidylate kinase variants pave the way to the rational design of modified human enzymes for the activation of other compounds applied in chemotherapy. In this line, the replacement of the immunogenic HSV1-TK enzyme, which is widely used in suicide gene therapy of certain cancers, by an engineered human nucleoside kinase capable of phosphorylating the prodrug ganciclovir selectively and more efficiently would be of prime importance (Bonini et al., 1997
; Cohen et al., 1999
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 August 2004;
accepted 15 November 2004.
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