The CRK3 protein kinase is essential for cell cycle progression of Leishmania mexicana

Molecular & Biochemical Parasitology 113 (2001) 189– 198 www.parasitology-online.com.

The CRK3 protein kinase is essential for cell cycle progression of Leishmania mexicana Paul Hassan 1, David Fergusson, Karen M. Grant, Jeremy C. Mottram * Wellcome Centre for Molecular Parasitology, Uni6ersity of Glasgow, Anderson College, 56 Dumbarton Road, Glasgow G11 6NU, Scotland, UK Received 10 August 2000; received in revised form 22 December 2000; accepted 4 January 2001

Abstract The Leishmania mexicana CRK3 gene encodes a cdc2-related protein kinase with activity towards histone H1. Attempts to disrupt both alleles of CRK3 in the promastigote life-cycle stage resulted in changes in cell ploidy, which were avoided only when an extra copy of CRK3 was expressed from an episome. This provides strong evidence that CRK3 is essential to L. mexicana. The cyclin-dependent kinase specific inhibitor flavopiridol inhibited affinity purified histidine tagged CRK3 (CRK3his) with an IC50 value of 100 nM and inhibited in vitro growth of L. mexicana promastigotes. Incubation of promastigotes with 2.5 mM flavopiridol for 24 h led to cell cycle arrest with an accumulation of 95% of cells in G2 or early mitosis (G2/M). Release from cell cycle arrest resulted in a semi-synchronous re-entry into the cell cycle; samples taken at 2, 4, and 6 h after release from the block were enriched for cells in G1 (68%), S-phase (70%), and G2/M phase (61%), respectively. This method of synchronisation was used to show that the majority of CRK3his activity towards the substrate histone H1 was present at G2/M. These data suggest that CRK3 has an essential role in controlling cell cycle progression at the G2/M-phase transition in L. mexicana promastigotes. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Leishmania; Cyclin-dependent kinase; Cell cycle; Flavopiridol

1. Introduction Investigations into the molecules that regulate the cell cycle of Leishmania during its life-cycle have identified two cdc2-related serine – threonine protein kinases that belong to the cyclin-dependent kinase (CDK) family, CRK1 and CRK3, [1 – 3]. Cyclin dependent kinases, such as cdc2 of Schizosaccharomyces pombe and CDC28 of Saccharomyces cere6isiae, are key regulators of the cell cycle and are ubiquitous throughout eukaryotes [4]. In both fission yeast and budding yeast, control of cell cycle progression is associated with the activity of a single CDK (cdc2 or CDC28, respectively) in a complex with G1 or G2-specific cyclins [5,6]. In mamAbbre6iations: CDK, cyclin-dependent kinase; CRK, cdc2-related kinase; FACS, fluorescence activated cell sorting. * Corresponding author. Tel.: + 44-141-3303745; fax: + 44-1413305422. E-mail address: [email protected] (J.C. Mottram). 1 Present address: Institut fuer Molekularbiologie und Biochemie, AG Molekulare Zellbiologie, Freie Universitaet Berlin, Fachbereich Humanmedizin, Hindenburgdamm 27, D-12203 Berlin, Germany.

malian cells, however, control of the G1/S and G2/M transitions requires two separate CDKs, cdk2 for G1/S progression [7] and cdk1 for the G2/M transition [8]. The leishmanial CRK1 protein kinase is encoded by an essential gene [9] and is post-translationally regulated in a stage-specific manner, being active in promastigotes and metacyclic promastigotes but not in amastigotes [1]. Several lines of biochemical evidence suggest that CRK3 is a functional cdk1 homologue. CRK3 forms the majority of the p13suc1 binding histone H1 kinase activity of L. mexicana, and associates with p12cks1, the Leishmania homologue of p13suc1 [2,10]. The leishmanial histone H1 kinase activity that binds p13suc1 is detected in the proliferative promastigote and amastigote stages but not in the cell-cycle arrested metacyclic stage [2]. In addition the L. major CRK3, which is 99% identical to L. mexicana CRK3, is capable of complementing for loss of function of cdc2 in a conditional mutant of Schizosaccharomyces pombe [3]. The role of CDKs as regulators of the cell cycle and the observation that their activity is frequently deregulated in tumour cells [11,12] initiated efforts aimed at

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identifying compounds that inhibit CDKs [13,14]. Such compounds have possible therapeutic use as anti-cancer agents and as treatments to control the proliferation of rapidly growing protozoan pathogens during infection [15]. These studies have led to the identification of a number of structurally unrelated chemical compounds that inhibit CDKs [16 – 19]. Although the chemical inhibitors of the CDKs are structurally diverse, many appear to inhibit kinase activity by the same mechanism, competitive binding to the ATP binding pocket. They differ however, in their target specificities. Some of the inhibitors are not specific to CDKs, and can inhibit a wide range of kinases e.g. staurosporine, which inhibits cdk1/cyclin B and protein kinase C (PKC) with an IC50 value of 3– 6 nM [20,21]. Butyrolactone I on the other hand displays good specificity against cdk1 and cdk2. It competes for ATP binding and inhibits the phosphorylation of cdk1 consensus sites of histone H1 and pRb [22]. One of the most interesting chemical inhibitors of CDKs is flavopiridol, a semi-synthetic flavone, structurally related to a natural alkaloid originally purified from the bark of Dysoxylum binectariferum, a plant indigenous to India [23]. Flavopiridol is a potent growth inhibitor of a number of tumour cell lines, and reduces the growth of human tumour xenografts in nu/nu mice [24]. Flavopiridol can inhibit cdk1 [25], cdk2 and 4 [26], and block mammalian cells in either G1 or G2 [27]. It can also affect S-phase progression in the protozoan parasite Plasmodium falciparum [28]. Flavopiridol is currently one of the few CDK inhibitors undergoing human clinical trials for the treatment of cancer [29]. In this study, Leishmania was subjected to genetic manipulation to show that CRK3 is essential to the parasite. In addition, the effects of the cyclin dependent kinase inhibitor, flavopiridol, on the activity of L. mexicana CRK3 and on cell cycle progression of in vitro cultured parasites were analysed. Results obtained indicate that L. mexicana CRK3 is an essential kinase controlling cell cycle progression at the entry into mitosis, and that currently available CDK inhibitors can inhibit CRK3 activity, reducing parasite growth rate. This indicates that CRK3 has potential as a novel drug target for the development of leishmanicidal drugs and that existing compounds may be possible leads for the development of more potent and specific CRK3 inhibitors.

2. Materials and methods

2.1. Leishmania mexicana Leishmania mexicana mexicana (MNYC/BZ/62/ M379) promastigotes were cultured at 25°C in

HOMEM medium as described earlier [1] supplemented with 10% heat inactivated foetal calf serum (FCS) (Labtech). Transgenic cell lines were grown in the presence of appropriate antibiotics at the following concentrations: hygromycin B (Boehringer Mannheim) 50 mg ml − 1, phleomycin 10 mg ml − 1 (Cayla, France) and G418 (Gibco BRL) 50 mg ml − 1. The cell line expressing histidine tagged CRK3 (CRK3his) with genotype (CRK3 /CRK3 [pXCRK3his]) has been described earlier [2].

2.2. Fla6opiridol block and release L. mexicana promastigotes were seeded at a density of 1× 106 cell ml − 1 and incubated in the presence of flavopiridol (a gift of Dr Swati Bal-Tembe, Hoechst Marion Roussel Ltd, Bombay, India). To release the flavopiridol block, cells were pelleted at 2000×g for 5 min and then washed twice with phosphate buffered saline (PBS) and once with fresh medium before being resuspended in an equal volume of fresh medium.

2.3. DNA content analysis by flow cytometry Mid-log phase L. mexicana promastigotes were harvested by centrifugation at 2000× g, for 5 min at 4°C. Cells were washed once in 10 ml PBS and then fixed by incubation in 70% methanol/30% PBS for 1 h at 4°C. Prior to analysis, fixed cells were harvested by centrifugation at 1000× g, for 10 min at 4°C, washed in 10 ml PBS and then resuspended in 1 ml PBS with RNAse A and propidium iodide at 10 mg ml − 1. The cells were incubated at 37°C for 45 min and then analysed using either a Becton Dickinson FACScalibur or a Coulter Epics/XL flow cytometer. Ten thousand cells were analysed for each sample. Cell cycle distribution was modelled using the ModFit LT software package (Verity Software House) in accordance with the standards detailed in [30].

2.4. Transfection The constructs used to disrupt CRK3 were based on those used earlier in our laboratory to delete the L. mexicana CPA gene [31]. The 5% and 3% CPA sequence from knockout plasmid pLMCPA-BLE [31] was replaced with CRK3 5% and 3% sequences (see Fig. 1A). Firstly, HindIII/SalI digestion of pGL89 [2] released a 1.1 kb fragment containing the 5% flank of CRK3, which was then cloned into the HindIII/SalI digested pLMCPA-BLE to form a CPA/CRK3 fusion construct. The CRK3 3% flank was generated by PCR using the following oligonucleotide primers; OL326 5%-GCAGATCTCCCGGGCAGTTGTTTGAGAT-3% and OL327 5%-GCAGATCTCCCGGGCAGTTGTTTGAGAT-3%.

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These primers contain engineered SmaI and BglII restriction sites. The PCR product was cloned into the pTAg vector (Ingenius) and the 339 bp SmaI/BglII insert subcloned into SmaI/BglII digested CPA/CRK3 fusion construct to produce the CRK3-BLE knockout construct, pGL97. The CRK3-HYG disruption cassette was generated by replacing the BLE drug resistance gene in pGL97 with the 1.0 kb SpeI/BamHI HYG fragment from pLMCPA-HYG [31] to give plasmid pGL105. The pGL100 construct for episomal expression of CRK3 was generated as follows, plasmid pGL89 was digested with EcoRI and HindIII and the resulting 1.0 kb fragment was gel purified and ligated to EcoRI/ HindIII digested pTEX vector [32]. Transfection of L. mexicana was performed according to the protocol of Coburn et al. [33] as described earlier [31,34]. 20 mg of circular plasmid or 10 mg of gel-purified linear plasmid DNA was added to cells that were immediately transfected by electroporation at 2.25

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kV cm − 1 with a Genepulser II (Biorad). After electroporation, cells were placed on ice for 10 min before being transferred to liquid medium (HOMEM). Cells were incubated overnight to allow expression of the drug selectable marker, before being plating on solid HOMEM/1% agar plates containing the appropriate antibiotics.

2.5. DNA manipulations DNA was prepared from 0.5 ml of late log-phase L. mexicana promastigotes by the mini-prep method [35]. For Southern blots, DNA was digested overnight with HindIII at 37°C. Fragments were separated by electrophoresis through a 0.8% agarose TBE gel and transferred to Nylon membrane following standard denaturation protocols as described earlier [9]. The membrane was probed under high stringency with a radiolabelled 2 kb HindIII fragment containing the

Fig. 1. Targeted gene disruption of CRK3. (A) A representation of the CRK3 locus and integrated targeting constructs. Changes in the size of the HindIII restriction fragment upon integration of either the CRK3-BLE or CRK3-HYG gene disruption cassettes are shown. (B) Southern blot analysis of wild type and CRK3 mutant cell lines. DNA was digested with HindIII, separated by agarose gel electrophoresis, transferred to membrane and hybridised with a radiolabelled 2.0 kb HindIII CRK3 gene fragment. Refer to Table 1 for genotypes. (C) DNA content analysis of wild type and CRK3 mutant cell lines.

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Table 1 L. mexicana mutant genotypes Clone

Drug resistance

Ploidy

Genotype

W583 W585 W625 W626 W638 W1187 W1188

Hyg Ble Hyg and Ble Hyg and Neo Hyg, Ble and Neo

Diploid Diploid Aneuploid Diploid Diploid

CRK3/CRK3::HYG CRK3/CRK3::BLE CRK3/CRK3/CRK3::HYG/CRK3::BLE CRK3/CRK3::HYG [pTEXCRK3] CRK3::HYG/CRK3::BLE [pTEXCRK3]

CRK3 gene, prepared by random primer labelling with the Prime-It II kit (Stratagene) according to the manufacturers instructions.

2.6. CRK3his histone H1 kinase assays About 1 × 108 cells were resuspended in 1 ml of LSGI (50 mM MOPS, pH 7.2; 100 mM NaCl; 0.1 mM EDTA; 0.1 mM EGTA; 10 mM NaF; 1 mM Na orthovanadate; 1% Triton X-100; 10% Glycerol with protease inhibitors, 1 mM 1,10-phenanthroline, 500 mg ml − 1 pefabloc SC, 100 mg ml − 1 leupeptin and 5 mg ml − 1 pepstatin A) and incubated on ice for 30 min. Samples were then centrifuged at 100 000× g for 45 min at 4°C and the supernatant removed (S-100 extract). Then 50 ml Ni-NTA agarose beads (Qiagen) were transferred to a 2 ml disposable plastic column (Pierce) and washed with 0.5 ml LSGI. The S-100 lysate was diluted 1:1 with 100 mM imidazole in LSG and incubated with the Ni-NTA agarose beads for 1 h at 4°C. The beads were then washed with the following regime; 5 ml LSG plus 50 mM imidazole, 5 ml HSLS (50 mM MOPS, pH 7.2; 500 mM NaCl; 0.1 mM EDTA; 0.1 mM EGTA; 10 mM NaF; 1 mM Na orthovanadate; 1% Triton X-100), and 5 ml LS-T (50 mM MOPS, pH 7.2; 100 mM NaCl; 0.1 mM EDTA; 0.1 mM EGTA; 10 mM NaF; 1 mM Na orthovanadate). Bound kinase was then eluted with 100 mM EDTA and then re-bound to p13suc1 beads as described earlier [10]. Beads were assayed for kinase activity as described [2] in the presence of a range of concentrations of flavopiridol. Samples were electrophoresed on a 12.5% SDS acrylamide gel, and the activity quantitated with a phosphorimager (Fuji).

3. Results

3.1. Targeted disruption of L. mexicana CRK3 We have shown earlier that CRK3 has many biochemical and sequence features in common with the cdc2 family of cyclin-dependent kinases, many of which are involved in cell cycle control [2]. To test whether CRK3 is essential to L. mexicana promastigotes, the

gene was targeted for disruption using targeting constructs based on those used earlier for gene disruption of the CPA [31] and CRK1 [9] genes. Two targeting constructs were created conferring resistance to hygromycin (pGL105) and phleomycin (pGL97) (Fig. 1A). Wild type L. mexicana promastigotes were transfected with 10 mg of gel-purified CRK3-HYG disruption cassette and transgenic clones were selected for hygromycin resistance. Names and genotypes of all clones are shown in Table 1. One of the heterozygote mutants (CRK3 /CRK3::HYG) isolated, clone W583, and wild type L. mexicana were transfected subsequently with the CRK3-BLE disruption cassette. Clones were selected either for resistance to phleomycin and hygromycin or phleomycin alone. Two double resistant clones (W625 and W626) and one phleomycin resistant clone (W585) were selected for further analysis. The genomic organisation of these selected clones was determined by Southern blot analysis using the 2.0 kb HindIII fragment, derived from the CRK3 locus, as a probe (Fig. 1B). A 2.0 kb HindIII fragment containing the wild type CRK3 gene (Fig. 1A) was detected in DNA prepared from wild type parasites (lane 1). In the DNA prepared from the W583 or W585 heterozygotes, either a 4.8 or a 4.1 kb fragment was detected, as well as the 2.0 kb fragment containing the CRK3 gene (lanes 2 and 3). These results indicate that in the hygromycinresistant heterozygote clone, W583, one allele of CRK3 has been successfully disrupted (CRK3 /CRK3::HYG). Likewise for the phleomycin-resistant heterozygote mutant, W585, (CRK3 /CRK3::BLE). Double resistant clones, W625 and W626, were found to contain the HYG and BLE resistance genes, as well as the 2.0 kb CRK3 fragment (lanes 4 and 5), indicating that two CRK3 alleles had been successfully disrupted with the drug resistance markers, but that the wild type CRK3 gene remained. The DNA content of the mutant cell lines was determined by fluorescence activated cell sorting (FACS) analysis of cells fixed and stained with propidium iodide (Fig. 1C). A normal diploid DNA content with two peaks corresponding to cells in G1 (2N DNA content) and G2 (4N DNA content) was observed for wild type cells (panel A). Similar patterns were observed for the heterozygote mutants, W583 and W585, (panels B and

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C). Clones W625 and W626, however, had approximately twice the normal diploid DNA content, with a G1 content of 4N and a G2 content of 8N, indicating that these cells are aneuploid (panels D and E). This change in ploidy has clearly occurred during the introduction of, and selection for, the second knockout construct, as was observed when the CRK1 gene was targeted for disruption [9]. The uneven nature of the DNA content histograms for the aneuploid clones, W625, and W626, with a less distinct separation into two clear peaks, suggests that there may be some variability in the DNA content of individual cells in the population and not all were tetraploid. To test the hypothesis that CRK3 is an essential gene and that the ploidy changes result from the selection for maintenance of at least one intact copy of CRK3, an attempt was made to express the gene from an episome prior to disrupting the second CRK3 allele. In this instance it should be possible to disrupt both alleles without causing any changes in ploidy, as the cells will still retain copies of the essential gene on an episome. CRK3 was cloned into the pTEX expression vector. The pTEX vector was designed for stable episomal expression in the related parasite, Trypanosoma cruzi [32] but has also been used to express a number of genes in Leishmania species [9,34,36]. The pTEXCRK3 (pGL100) plasmid was introduced by electroporation into the W583 cell line and cells were selected for resistance to 50 mg ml − 1 Geneticin (G418) and 50 mg ml − 1 hygromycin. One of the resultant clones, W638, was selected for further study and shown by Southern blot analysis to contain episomal copies of the CRK3 gene and one remaining wild type CRK3 allele (Fig. 1B, lane 6). The CRK3-BLE targeting fragment was then transfected into the W638 heterozygote mutant (CRK3 / CRK3::HYG [pTEXCRK3]), with transfectants being selected in the presence of 50 mg ml − 1 hygromycin and 10 mg ml − 1 phleomycin but in the absence of Geneticin. This selection protocol was chosen to avoid problems associated with the triple drug selection. Two clones (W1187 to W1188) were selected for analysis and were both found to be resistant to Geneticin, as expected if the pTEXCRK3 plasmid had been introduced into, and retained within, the cell lines. W1187 to W1188 were found to have a normal diploid DNA content (Fig. 1C, panel 7 and 8). Southern blot analysis (Fig. 1B) showed that both W1187 and W1188 lacked the 2.0 kb wild type CRK3 fragment but did contain the 4.1 kb fragment indicative of the correct integration of the CRK3BLE targeting construct (lanes 7 and 8). These clones were also found to contain multiple copies of the pTEXCRK3 episome (intensely hybridising fragments of 5 –10 kb DNA in Fig. 1B, lanes 7 and 8). These results suggest that these two clones lack a genomic copy of CRK3 due to disruption of both alleles, and that CRK3 is being expressed from the pTEX episome.

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3.2. Fla6opiridol inhibits CRK3his and the growth of L. mexicana promastigotes CRK3his protein was purified by Nickel NTA affinity chromatography from a transgenic L. mexicana cell line that expresses histidine-tagged CRK3 [2]. Histone H1 kinase activity of CRK3his was assessed in the presence of increasing concentrations (1 nM –100 mM) of flavopiridol to determine the IC50. Relative kinase activity was assessed by phosphorimage analysis (Fig. 2A) and the plotted results give an IC50 value of 100 nM (80 and 130 nM in two other experiments). This value is comparable to IC50 values for a number of human CDKs, which range from 100 to 400 nM [25,26]. These results indicate that flavopiridol is a potent inhibitor of CRK3his in vitro. To test the effect of flavopiridol on L. mexicana, promastigotes were seeded in triplicate at a density of 1.0× 106 cell ml − 1 and incubated in the presence of a range of concentrations of the drug. Cells were counted at 24 h intervals and the mean value of triplicate cell counts was plotted (Fig. 2B). Growth of Leishmania was inhibited by flavopiridol in a dose dependent manner. A concentration of 1.0 mM resulted in complete arrest of promastigote growth, whereas lower concentrations only partially inhibited cell growth and higher concentrations resulted in cell death. About 50% inhibition of cell growth was achieved at a concentration of approximately 250 nM. Attempts to test the effect of flavopiridol on amastigote growth in explanted mouse macrophages in vitro failed due to the high level of toxicity of the drug to the mammalian cells.

3.3. Fla6opiridol causes G2 -phase cell cycle arrest Given that CRK3 is a putative controller of cell cycle progression and is inhibited in vitro by flavopiridol, then it is probable that the inhibition of promastigote growth caused by flavopiridol is due to disruption of normal cell cycle progression. L. mexicana promastigotes were seeded at a density of 1× 106 cell ml − 1 in the presence of 0, 1.0, 2.5 or 5.0 mM flavopiridol. Samples were taken at various time points and analysed by FACS to determine the overall DNA content (Fig. 3). Cells grown in the absence of flavopiridol showed a normal cell cycle distribution (Panels A–D). Treatment with 2.5 mM flavopiridol led to an accumulation of cells with a 4N DNA content after incubation for 12 (Panel J), 18 (Panel K) or 24 h (Panel L). Microscopic examination of arrested cells stained with DAPI showed that the cells had not undergone mitosis as they contained one nucleus and 1 kinetoplast. These cells must be arrested in the G2 phase or early M-phase of the cell cycle, after DNA replication, but before cell division. The lack of reliable markers for different stages of the Leishmania cell cycle prevent a more detailed character-

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isation of the cell cycle block at this time. Cells blocked with flavopiridol for a longer time period (\ 24 h), or at higher concentrations of flavopiridol, began to die, resulting in a wider spread of fluorescence intensity signals as their nuclei broke apart (e.g. Panel P).

3.4. Release from fla6opiridol inhibition leads to semi-synchronous progression through the cell cycle To test whether the G2/M phase arrest caused by flavopiridol was reversible, log phase L. mexicana promastigotes were incubated in the presence of 2.5 mM flavopiridol, washed to remove the drug and then allowed to recover. Samples taken at various time points were prepared for FACS analysis to determine their position within the cell cycle (Fig. 4). At the 12 h time point, the sample showed the normal cell cycle distribution of logarithmically growing promastigotes. At time 0 h, 95% of cells were blocked in G2. Following release from growth arrest, FACS analysis showed a semi-synchronous re-entry into the cell cycle with 65% of cells in the G1 phase of the cell cycle 2 h after release from the block, with only 26% remaining in G2. After a further 2 h, 70% of cells were in S-phase, whilst 12 and 18% were in G1 and G2 phase, respectively. At the 6 h time point the majority of cells, 61%, were in G2, whilst 21 and 18% were in G1 and S-phase, respectively. Although release from flavopiridol inhibition is not completely synchronous, this method can be used to enrich samples for cells in specific cell cycle stages. Samples taken at 2, 4, and 6 h after release from the block were enriched for cells in G1 (68%), S-phase (70%), and G2 phase (61%), respectively.

3.5. L. mexicana promastigotes released from cell cycle arrest show fluctuating CRK3his acti6ity, peaking in G2

Fig. 2. Flavopiridol inhibits CRK3his and prevents the growth of L. mexicana promastigotes. (A) CRK3his was purified by nickel-NTA agarose affinity selection from L. mexicana cell line CRK3 /CRK3 [pXCRK3his]. Histone H1 kinase activity was assayed in the presence of increasing concentrations of flavopiridol, and the relative kinase activity was assessed by phosphorimaging analysis. Results are plotted as a percentage of uninhibited kinase activity. (B) L. mexicana promastigotes were seeded at a density of 1 × 106 cell ml − 1 and incubated in the presence of flavopiridol. Cell density was determined at 24 h intervals and the mean result of triplicate values is plotted.

To analyse the activity of CRK3 upon release from cell cycle inhibition by flavopiridol, log phase L. mexicana promastigotes expressing CRK3his were subjected to the same block and release protocol as described for wild type promastigotes. Cells were collected at various time points over a 28 h period, after which they were prepared for FACS analysis and assayed for CRK3his kinase activity (Fig. 5). At the 12 h time point, cells were approximately evenly distributed between the three cell cycle compartments. At time 0 h, however, after incubation with flavopiridol, 70% of cells were blocked in G2. FACS analysis showed a semi-synchronous re-entry into the cell cycle, with an increase in the proportion of cells in G1 relative to G2 within the first 6 h post release. A peak in G2/M occurs at about 10 h post release and corresponds to a similar trough in G1. The activity of CRK3his kinase was found to coincide with the G2/M peak. A second G1 peak at 18 h and a G2 peak at 24 h can be observed, coincident with an increase in CRK3his activity, however the cells are becoming asynchronous by this time. These results would suggest a cell cycle time of about 10–12 h for L. mexicana, which agrees with the observed doubling times in culture.

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Fig. 3. DNA content analysis of cells incubated with flavopiridol. FACS assessment of the DNA content of L. mexicana promastigotes incubated in the presence of flavopiridol. Cells were fixed, stained with propidium iodide, and 10 000 cells were counted on a Coulter Epics/XL flow cytometer.

4. Discussion In this study, we provide evidence that CRK3 is essential for cell cycle progression of L. mexicana. Attempts to generate CRK3 null mutant cell lines in which both copies of CRK3 were disrupted by the site-specific integration of a gene-targeting construct invariably failed. One allele of CRK3 could be deleted reproducibly with equal efficiency by homologous integration of either the CRK3-HYG or CRK3-BLE gene disruption constructs (Fig. 1). However, attempts to integrate the second targeting construct, in order to generate a null mutant, resulted in ploidy changes in the resultant transfected cell lines (Fig. 1 Panel C). The mechanism by which such changes in ploidy occur upon gene targeting is unknown at present, but this phenomenon is generally accepted as positive confirmation that the gene being targeted for disruption is essential [9,37,38]. If the targeted gene is essential, then it is not possible to obtain double-antibiotic resistant mutants unless ploidy changes, or a genomic rearrangement, have allowed the double resistant cells to retain at least one copy of the targeted gene [9,37,39,40]. Alterations in ploidy do not occur if extra-chromosomal CRK3 is introduced into the parasite prior to disruption of the second native CRK3 allele, showing that the ploidy phenotype is due to a requirement to maintain a copy of CRK3, rather than a by-product of

the transfection protocol. This was validated further by the observation that there was no need to select for the Neo-containing episome in the allelic null mutants, as only those cells that retained the episome and integrated the second targeting fragment were able to grow. The finding that CRK3 is essential led us to test known CDK inhibitors for their effects on the enzyme and on the parasite itself. Flavopiridol was one of the inhibitors chosen as it is a potent CDK inhibitor and has reached clinical trials as an anti-cancer drug [29]. Studies on the effects of flavopiridol may, therefore, prove useful in determining the structural requirements for selective inhibition of CRK3, leading to the design of lead compounds that could be developed further as anti-leishmanial drugs. Flavopiridol inhibited purified CRK3his activity and killed promastigotes grown in vitro, however the toxicity of the compound precluded a study on its effects on amastigotes in macrophages. One interesting outcome of the study was the finding that flavopiridol arrested L. mexicana in the G2/M phase of the cell cycle. This inhibition could be effectively reversed, leading to semi-synchronous progression through the next complete cell cycle, after which synchrony was lost. The ability to block and release cell cycle progression using flavopiridol enables the collection of sample populations enriched for cells in a particular cell cycle phase. This may prove useful in identifying cell-cycle regulated mRNAs, proteins or en-

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zyme activities in L. mexicana. This method was used to analyse the fluctuating activity of the CRK3his kinase throughout the post-release cell cycle. The initial peak of kinase activity, at the blockade point, is most likely due to the fact that the cells are blocked in the cell cycle compartment when CRK3 is most active, G2/M. In the presence of flavopiridol, CRK3his is presumably inactivated, as it is in vitro, by competitive inhibition of ATP binding. However, during affinity purification of the enzyme the flavopiridol would be released from the ATP binding site, resulting in restoration of CRK3his kinase activity. The cycling of CRK3his activity ‘in phase’ with G2/M and ‘antiphase’ with G1 through the initial cell cycles after release from the flavopiridol cell cycle block, argues that CRK3his

Fig. 5. CRK3his kinase activity fluctuates during the cell cycle. The CRK3 /CRK3 [pXCRK3his] cell line was incubated in the presence of 2.5 mM flavopiridol for 12 h. Cells were then washed and resuspended in fresh medium. Samples were removed at various time points for determination of cell cycle distribution and CRK3his kinase activity. (A) Cell cycle distribution. (B) Quantification of relative histone H1 kinase activity by phosphorimaging.

Fig. 4. Release from flavopiridol inhibition leads to semi-synchronous progression through the cell cycle. Wild type L. mexicana promastigotes were incubated in the presence of 2.5 mM flavopiridol for 12 h. Cells were then washed and resuspended in fresh medium (time 0). Samples were removed at various time points for determination of cell cycle distribution by FACS analysis. (A) FACS profiles of cells before, during and after flavopiridol incubation. (B) Cell cycle distribution as determined using the ModFit LT software package (Verity software house).

demonstrates the expected timing of activity for a G2/ M cyclin-dependent kinase. In this respect the Leishmania CRK3 differs from S. cere6isiae CDC28, which is active at both G2/M and G1/S boundaries [41], but shows similarity to the mammalian CDK1, which is active at the G2/M boundary [42]. It should be noted that CRK3 may be active at G1/S, but bound to a different cyclin, producing a complex that is not inhibited by flavopiridol to the same extent, or that does not phosphorylate histone H1. Mammalian CDK2 complexes display different sensitivities to two other CDK

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inhibitor compounds; purvalanol B and kenpaullone, depending on the cyclin partner [43,44]. Such differences however, have not been reported for flavopiridol. Although the growth of the L. mexicana CRK3his expressing cell line used in this study was found to be inhibited by flavopiridol to the same extent as wild type cells (data not shown), there is nevertheless a difference in the cell cycle distribution of cells incubated with 2.5 mM flavopiridol for 24 h, 70% blocked in G2 as opposed to 95% in wild type cells exposed to the same treatment. This difference may be a consequence of the fact that the CRK3his expressing cell line contains CRK3his in addition to normal levels of CRK3. The growth rate of this line, however, resembles that of wild type cells, probably because the overall levels of active kinase complex are not increased by expression of CRK3his, as cyclin binding is likely to be the limiting factor in formation of an active kinase complex. Differences in the cell cycle profile between the CRK3his expressing cell line and wild type cells incubated with flavopiridol strengthen the argument that CRK3 is a primary target for flavopiridol inhibition. In summary, this study provides evidence that CRK3 has biochemical features that suggest a role in controlling the G2/M transition and thus has functional homology to cdc2. Gene knockout experiments show that CRK3 is essential to the parasite and that flavopiridol, a potent inhibitor of CRK3, is lethal to Leishmania. These data show that CRK3 is a promising target for rational drug design. A large number of chemical CDK inhibitors have been synthesised [14] and we are now in the process of screening a collection of these for activity against CRK3 in vitro and in vivo.

Acknowledgements The Medical Research Council of the United Kingdom and the Robertson Trust supported this work. JCM is a MRC Senior Research Fellow.

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