Cancer Therapy Vol 2, 389-402, 2004

Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, GPO Box 2100, Adelaide South Australia 5001, Australia.

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*Correspondence: Professor GJ Barritt, Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, GPO Box 2100, Adelaide South Australia 5001, Australia; Telephone: (+61 8) 8204 4260; Fax: (+61 8) 8374 0139; E-mail: Greg.Barritt@flinders.edu.au

Key words: TRPL, Ca²⁺ channels, prostate cancer cells, Tet-On promoter, doxycycline

Abbreviations: 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide, (MTT); cytoplasmic Ca²⁺ concentration, ([Ca²⁺]_cyt); phosphate-buffered saline, (PBS); transient receptor potential, (TRP); transient receptor potential-like, (TRPL)

The development, growth and function of prostate epithelial cells are androgen dependent. Androgens elicit these effects via the androgen receptor (Isaacs et al, 1992; Colombel et al, 1996; Denmeade and Isaacs, 1996; Gschwend, 1996). Tumorigenic prostate epithelial cells exhibit uncontrolled cell growth which, in the early stages of the disease, is dependent on the presence of androgen. Any manipulation (e.g. androgen ablation) which reduces the concentration of androgen initiates apoptotic cell death in normal prostate epithelial cells and in prostate cancer cells. In early stage prostate cancer, androgen ablation leads to regression of the disease. However, androgen-resistant clones of prostate epithelial cells often arise. These cells do not undergo apoptosis so that androgen-independent tumour cells proliferate and metastasise (Isaacs et al, 1992; Colombel et al, 1996; Denmeade and Isaacs, 1996; Gschwend, 1996). There is presently no effective way of killing androgen-independent tumour cells in prostate cancer patients (Isaacs et al, 1992; Colombel et al, 1996; Denmeade and Isaacs, 1996; Gschwend, 1996).

The molecular mechanisms by which androgen ablation induces apoptosis in androgen-sensitive prostate cancer cells are not yet fully defined (Isaacs et al, 1992; Colombel et al, 1996; Denmeade and Isaacs, 1996; Gschwend, 1996). However, several studies suggest that apoptosis caused by androgen ablation is associated with a sustained increase in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) (Martikainen et al, 1991; Steinsapir et al, 1991; Isaacs et al, 1992; Tombal et al, 1995; Denmeade and Isaacs, 1996; Gutierrez et al, 1999). Sustained high [Ca²⁺]_cyt leads to cell toxicity and death through a variety of mechanisms in which the accumulation of Ca²⁺ in mitochondria and subsequent release of mitochondrial metabolites and proteins play major roles (McConkey and Orrenius, 1997; Porn-Ares et al, 1998; Kass and Orrenius, 1999; Jambrina et al, 2003). Thapsigargin (an inhibitor of the endoplasmic reticulum (Ca²⁺ + Mg²⁺)ATPase) has been shown to induce apoptosis in both androgen-sensitive and androgen-insensitive prostate cancer cell lines (Furuya et al, 1994). The apoptotic effects of thapsigargin are associated with a decrease in the concentration of the Ca²⁺ in the endoplasmic reticulum and, as a consequence of this, sustained Ca²⁺ inflow through store-operated Ca²⁺ channels in the plasma membrane (Furuya et al, 1994; Skryma et al, 2000; Putney et al, 2001). While the mechanism by which thapsigargin induces cell death is complex (it may involve a decrease in concentration of Ca²⁺ in the endoplasmic reticulum and other signalling pathways as well as a sustained high [Ca²⁺]_cyt), the increase in [Ca²⁺]_cyt appears to play a key role in the induction of apoptosis (Furuya et al, 1994; McConkey and Orrenius, 1997; Porn-Ares et al, 1998; Kass and Orrenius, 1999; Skryma et al, 2000).

A sustained high [Ca²⁺]_cyt can also be induced in cells by the heterologous expression of constitutively active Ca²⁺-permeable channels in the plasma membrane. For example, it has recently been shown that Ca²⁺ inflow through TRPM7 channels induces neuronal cell death in oxygen-glucose deprivation (Aarts et al, 2003) and through TRPV1 channels, the death of Jurkat cells transiently-transfected with TRPV1 (Jambrina et al, 2003). We have recently shown that heterologous expression of the Drosophila melanogaster TRPL (transient receptor potential like) non-selective cation channel (Phillips et al, 1992) in the androgen-sensitive LNCaP cell line induces Ca²⁺ inflow and cell death (Zhang et al, 2003). In these experiments, expression of the TRPL protein was placed under the control of the constitutive CMV promoter. However, in developing this system for the potential treatment of prostate cancer, it would be highly desirable to be able to control the amount and timing of expression of the TRPL protein. Therefore, the aim of the present experiments was to develop an inducible system for the regulated expression of TRPL and hence for the controlled killing of prostate cancer cells by a sustained increase in [Ca²⁺]_cyt. The experiments have been conducted with the PC-3 androgen-insensitive prostate carcinoma cell line (derived from metastatic tumour tissue obtained from the lumbar vertebra of a 62 year old patient) (Webber et al, 1997). The Tet-On (tetracycline-controlled transcription activation system activated by the tetracycline derivative, doxycycline (Gossen et al, 1993, 1995; Gossen and Bujard, 1995)) was used as the prototype inducible expression system. This has been successfully employed in the controlled expression of a number of different proteins in a variety of mammalian cell lines (Gossen et al, 1993, 1995; Gossen and Bujard, 1995). The results reported here show that the Tet-On tetracycline expression system can be effectively used to control the expression of TRPL in PC-3 cells. The consequences of increased TRPL expression include enhanced Ca²⁺ inflow and increased [Ca²⁺]_cyt and cell death by apoptosis. These results provide an "in principle" demonstration that inducibly-controlled TRPL expression can provide controlled killing of prostate cancer cells.

PC-3 prostate cancer cells and pBluescript SK(+), containing the Drosophila Trpl cDNA insert, were kindly provided by Professor Wayne Tilley, Hanson Research Institute, Adelaide and Dr. Leonard E. Kelly, Department of Genetics, University of Melbourne, Melbourne, respectively. Opti-MEMâ and LipofectAMINE^TM2000 were obtained from GibcoBRL (Life Technologies, Melbourne, Australia); EDTA, doxycycline, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), fura-2 acetoxymethyl ester (Fura-2/AM) and pluronic F-127 from Molecular Probes (Eugene, OR, USA); FuGENE^TM 6 transfection reagent from Roche Applied Science (Castle Hill, NSW, Australia); plasmids for the Tet-On gene expression system (pTet-On, pTRE-Luc, pTK-Hyg, pBI-L and pTet-tTs) from Clontech (Palo Alto, Ca, USA); luciferase assay kit from Promega (NSW, Australia); PCR primers and pUC DNA markers from Geneworks Pty Ltd (South Australia).

PC-3 cells were routinely grown in RPMI-1640 medium supplemented with 5% (v/v) foetal bovine serum (FBS), 100 U/ml penicillin and 100 m g streptomycin (complete RPMI-1640 medium) (Tilley et al, 1995). PT-20-1 (Tet-On PC-3) cells were routinely grown in complete RPMI-1640 medium supplemented with 100 m g/ml G418. MP25 and MP40 cells (Tet-On PC-3 cells harbouring the TRPL and luciferase genes) were routinely grown in complete RPMI-1640 medium supplemented with 100 m g/ml G418 and 25 m g/ml hygromycin.

The plasmids pBluescript-Trpl, pTet-On, pTRE-Luc, pTK-Hyg, pBI-L, pBI-L-Trpl and pTet-tTS were transformed into competent bacterial cells (E. Coli XL1-Blue) by electroporation (Bio-Rad Gene Pulser) at 1.8 kV, 25 m F, and propagated using standard procedures. TRPL cDNA was subcloned from pBluescript SK(+) (Stratagene) into the pBI-L response plasmid using the Not I and Hind III restriction enzymes sites. PCR amplification was carried out to ensure that the full length of Trpl cDNA was successfully ligated into the pBI-L-Trpl plasmid and that the 5'-ends of the Trpl cDNA was located downstream of the minimal CMV promoter.

Electroporation of PC-3 cells (Bio-Rad Gene Pulser) was performed at 500 volts and 25 m F. For transfection of PC-3 cells using FuGENE^TM6, cells (5 x 10⁵) were plated in 2 ml RPMI-1640-5% (v/v) FBS in a 35 mm culture dish and incubated until the cell density was at 50-80% confluency (48 h). FuGENE6/plasmid DNA complexes were prepared by adding a mixture of FuGENE6 (3 m l) and RPMI-1640 (serum free) (97 m l) into diluted plasmid DNA (10-1000 ng) and incubating at room temperature. After 15 min, the FuGENE6/plasmid DNA mixture was added into each 35 mm culture dish dropwise, and the cells incubated at 37^oC in 5% CO₂/95% O₂ (v/v). For transient-transfection of PC-3 cells using LipofectAMINE^TM 2000, cells (5 x 10⁵) were plated in 2 ml RPMI-5% (v/v) FBS in a 35 mm culture dish and incubated until the cell density was at 90-95% confluency (72 h), according to the manufacturer's instructions. DNA-LF2000 was prepared by combining the diluted plasmid DNA (1 m g in 200 m l Opti-MEMâ ) with lipofectamine (3 m l in 200 m l Opti-MEMâ ) and incubating at room temperature for 20 min. The DNA-LF2000 reagent complex was then added directly to each culture dish, mixed by rocking, and the cells incubated at 37° C. After 4 h, the medium was replaced with fresh RPMI-5% (v/v) FBS.

The isolation of adherent cell clones from cells grown in 35 mm culture dishes was conducted using 3-5 mm² filter paper disks (Whatman Number 3) (Domann and Martinez, 1995). The cells on the filter paper were placed into a single well of a 24 well-plate containing 1 ml of complete RPMI-1640 medium supplemented with G418 or G418 plus hygromycin at the concentrations indicated in the text and legends to figures. The 24 well-plates were incubated in CO₂ (5% in O₂) at 37° C until the cells in each well (derived from a single isolated cell) had reached confluency. The cells were harvested by trypsinization, subcultured into 25 cm² culture flasks containing complete RPMI-1640 medium supplemented with G418 and/or G418 plus hygromycin at the concentrations indicated, and the cells grown to near confluency. A sample of each clone was frozen in liquid N₂.

PC-3 cells were transfected with the pTet-On plasmid by electroporation. The transfected PC-3 cells (3 x 10⁴ cells/ml, 50 mm culture dish) were grown in the presence of 200 m g/ml G418 to allow for the selection of cells containing pTet-On. After 5 weeks in selective culture medium, 30 healthy and fast-growing G418-resistant cell colonies were isolated. To determine which of these expressed the rTetR/VP16 transcription activator (regulates gene expression from the pTRE-Luc plasmid), each isolated G418-resistant cell line was then transiently-transfected with 10 ng pTRE-Luc and grown at 1.5 x 10⁴ cells/ml (35 mm culture dishes). After 48 h incubation in the absence, or presence, of 1 m g/ml doxycycline, cell extracts were prepared and analysed for luciferase activity. Out of the 30 G418-resistant clones isolated, three exhibited the highest degree of doxycycline-induced luciferase reporter activities (24-, 12-, and 19-fold). One (PT-20-1), which exhibited the highest induction of luciferase in response to doxycycline and the lowest background luciferase activity in the absence of doxycycline, was chosen as the Tet-On PC-3 cell line to be used for subsequent transient expression of TRPL and for the generation of double-stable Tet-On PC-3 cells containing Trpl cDNA.

Development of the inducible Tet-On system for expression of the TRPL gene in PC-3 cells required two-consecutive stable transfections (Gossen et al, 1995). The first is to introduce the Tet-On plasmid, which contains the regulatory gene encoding the transcription activator rtetR/VP16 under the control of a CMV promoter. The second is to introduce the TRPL gene under the control of the TRE-element and a minimal CMV promoter. The TRE-element is activated when it binds to rtetR/VP16 in a doxycycline dependent manner (Gossen et al, 1995). This would be expected to promote expression of the TRPL gene.

To generate double-stable PC-3 cell lines inducibly expressing both TRPL and luciferase, PT-20-1 Tet-On PC-3 cells (PC-3 cells stably-transfected with the Tet-On plasmid) were co-transfected with a given amount (100, 200, 600, 1000 or 2000 ng) of the pBI-L-Trpl plasmid (1000 ng) (which contains cDNA encoding the luciferase and TRPL proteins under the control of a bi-directional promoter) plus the pTK-Hyg (400 ng) plasmid, and grown in the presence of hygromycin (50 m g/ml) and G418 (100 m g/ml). All the individual hygromycin-resistant cell lines obtained from the transfections using different amounts of pBI-L-Trpl grew slowly, and many cells exhibited polymorphic morphology. A large number of dead cells was observed in each transfected cell line. (PT-20-1 cells transfected with the pBI-L plasmid (controls) grew at the same rate as untransfected PT-20-1 cells and exhibited normal morphology.)

Since the cells which survived the co-transfection with pBI-L-TRPL and pTK-Hyg were unstable and polymorphic, and did not exhibit doxycycline-inducible luciferase, a second subcloning of those cells which survived from the 1000 ng pBI-L-TRPL transfection was carried out in an attempt to obtain a stable and morphologically-homogeneous cell line expressing TRPL under control of the Tet-On promoter. Surviving cells were cultured in the presence of a higher concentration of hygromycin (150 m g/ml) (in order to increase the selection pressure) while maintaining the concentration of G418 at 100 m g/ml. After 8 weeks, a number of cell-colonies were isolated. Twenty of these individual isolated clonal cell lines were grown in the presence of 150 m g/ml hygromycin and G418 (100 m g/ml) for a further two passages, then maintained in the presence of a low concentration of hygromycin (25 m g/ml) plus G418 (100 m g/ml). These cells were tested for Trpl gene incorporation by PCR amplification using the FBTRPL2 / RBTRPL2 primers. A PCR band of 336 bp was obtained from each of 9 clones, indicating that the 5'-end of the Trpl gene had been incorporated into each clone. PCR amplification using the C20A/pBI-A1 primers, which amplify the 3'-end of the TRPL gene, was performed to further confirm incorporation of the TRPL gene in these cell lines. A predicted 1.5 kb fragment was obtained for all 9 clones, confirming that the Trpl gene had been stably incorporated into each of these cell lines. To test for incorporation of the luciferase gene, PCR amplification was carried out using the FLUC/RLUC primers. A predicted 280 bp fragment was obtained from all 9 clones, indicating that the luciferase gene had also been incorporated into each cell line.

Two (MP25 and MP40) of the 9 clones described above showed the highest doxycycline induction of luciferase and were chosen for further study. When grown in early passages, MP25 and MP40 showed a high luciferase activity in the absence of doxycycline and a 3- and 2.5-fold induction, respectively, of luciferase following incubation with doxycycline (1 m g/ml for 48 h) (c.f. the amount of luciferase induction observed in other cell types (Isaacs et al, 1992; Colombel et al, 1996; Denmeade and Isaacs, 1996; Gschwend, 1996). (However in later passages, no luciferase induction was observed, even when a range of doxycycline concentrations was employed.) In order to confirm that the synthesis and action of the rTetR/VP16 transcription factor were effective in MP25 and MP40 cells, PC-20-1, MP25 and MP40 cells were each transiently-transfected with the pTRE-Luc plasmid In each case a 10- to 15-fold induction of luciferase by doxycycline was observed, thus indicating that the rTetR/VP16 transcription factor was active in these cells.

To amplify Trpl cDNA in the original or reversed orientation in the pBluescript-Trpl plasmid, the primer pairs C20A/RSP or C20A/USP were employed. RSP (5'-GGAAACAGCTATGACCATG-3') (antisense) and USP (5'-GTAAAACGACGGCCAGT-3') (sense) are two primers that flank the multiple cloning site of pBluescript. The C20A sense primer (5'-AGTGGAAGTTTGCCCGAACC-3') corresponds to a short sequence within Trpl (2195-2214) (Phillips et al, 1992). The predicted size of the amplified product fragments for each pair C20A/RSP and C20A/USP is approximately 1.5 kb.

To amplify Trpl cDNA in the pBI-L-Trpl plasmid and in the PC-3 cells incorporating the Trpl gene, three PCR primer pairs were designed. These were FETRPL1 (5'-AACACTCGTGCCTCAGATGG-3') (sense) and RETRPL1 (5'-TCCCCAAACTCACCCTGAAG-3') (antisense), which amplify the 3'-end of the TRPL DNA sequence (Zimmer et al, 2000) to yield a product of 354 bp; C20A (sense) and pBI-L A1 (5'-AGAGATATCGTCGACAAG-3') (antisense), which amplify the 3' half of the TRPL DNA sequence (Zimmer et al, 2000) to yield a product of approximately 1.5 kb; and FBTRPL2 (5'-CCATCCACGGTCTTTTGACC-3') (sense) and RBTRPL2 (5'-CGTCGGCAGCTTCTTTTTGC-3') (antisense) which amplify the 5'-end of the TRPL DNA sequence and the P_CMV-1 promoter in the pBI-L-TRPL plasmid, to yield a product of approximately 336 bp.

To amplify a region of the luciferase gene in the pBI-L-TRPL plasmid and in the luciferase gene incorporated in PC-3 cells, primers were designed to amplify a sequence of the P_CMV-2 promoter and luciferase cDNA. The primers were FLUC (5'-TAGCTTCTGCCAACCGAACG-3') (sense) and RLUC (5'-CCATCCACGCTGTTTTGACC-3') (antisense), which yield a product of 280 bp.

For PCR amplification of TRPL and luciferase genes in plasmids, plasmid DNA was purified using the QIAGEN QIA filter Plasmid Midi kit according to the manufacturers instructions. Bulk PCR reaction mixtures contained 2 mM MgCl₂, 100 m M of each dNTP, 0.4 m M of each primer (sense, antisense), and 0.4 units Taq polymerase in 1x Taq PCR buffer. Each reaction mixture (23 m l) was overlaid with mineral oil then 2 m l of plasmid template DNA (diluted in sterile H₂O) added. The thermal profile for PCR amplification (Hybaid Omn-E PCR machine) consisted of 1 cycle of 94° C for 5 min, 40 cycles at 94° C for 1 min (denaturation), at 55° C for 1 min (annealing), at 72° C for 1 min (extension), 1 cycle at 72° C for 5 min, and 1 cycle at 15° C for 1 min (completion). Amplified products were visualized (ethidium bromide) after separation on 1% agarose.

For PCR amplification of regions of the Trpl and luciferase genes incorporated in PC-3 cells, the cells were harvested using 0.1% (w/v) trypsin/EDTA, and washed once with 1 ml PBS. 50 m l of cell lysis buffer (50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.1 mg/ml gelatin, 0.45% (v/v) Nonidet P40, 0.45% (v/v) Tween-20 and proteinase-K (6 m l of 10 mg/ml added just before use) was added to each pellet, and the mixtures incubated at 60° C for 1 h, then at 95° C for 15 min to denature enzyme activity. The resulting cell-lysate was used for PCR amplification.

Luciferase activity was measured using a Promega Luciferase Assay kit, according to the manufacturer's instructions.

A polyclonal rabbit anti-TRPL antibody was raised (Zhang et al, 2003) against a synthetic peptide (fused with diphtheria toxin) corresponding to the 14 carboxy terminal amino acids of the Drosophila TRPL protein. The peptide sequence, which included a cysteine residue at the amino terminus to facilitate coupling to the carrier diphtheria toxoid, was CDSNFDIHVVDLDEK (Niemeyer et al, 1996). Serum from the fifth boost of peptide injection was used to precipitate IgG (using cold half-saturated ammonium sulfate). The precipitated protein was sedimented by centrifugation at 15,000 rpm at 4° C for 30 min and resuspended in PBS, dialysed twice at 4° C in PBS, then purified by affinity chromatography on immunizing peptide bound to epoxy-activated sepharose 6B (Pharmacia). The adsorbed antibody was eluted with 4.9 M MgCl₂. The specificity of the resulting anti-TRPL antibody was assessed by Western blot analysis using a Drosophila head-extract as a source of the TRPL protein.

Whole cell lysates were prepared by adding 100 m l of lysis buffer (1% (v/v) Triton-X100, 0.1% (w/v) SDS, 10 mM leupeptin, 5 mM pepstatin, 10 mM PMSF and 0.001% (v/v) b -mercaptoethanol in PBS, pH 7.4) to the cell pellet (1 x 10⁶ cells), mixing the suspension by pipetting, maintaining the suspension for 30 min at 4° C, centrifuging (1,000xg for 5 min at 4° C) and collecting the supernatant.

Crude membrane extracts were prepared by adding 500 m l of 5 mM Tris-HCl, (pH 7.5), containing 5 mM EDTA, 10 mM leupeptin, 5 mM pepstatin, 10 mM PMSF and 0.0002% (v/v) b -mercaptoethanol to the cell pellet (1 x 10⁶ cells), lysing the cells at 4° C (by injecting the cell suspension through a 25-guage syringe needle 5-10 times), centrifuging (1,000xg for 5 min at 4° C), transferring the supernatant to a new tube, and centrifuging at 16,000xg for 30 min 4° C, and collecting the pellet. The pellet was then resuspended in 50 m l of 5 mM Tris-buffer and the solution stored at -20° C. Protein concentrations were measured using the Bradford method (Bradford and Bradford, 1976) with bovine serum albumin (Sigma) as a standard.

Western blot analysis was performed as described previously (Zhang et al, 2003), using rabbit polyclonal anti-TRPL antibody (1:100 dilution at 4° C overnight) and horse-radish peroxidase-conjugated anti-rabbit IgG (1:100 dilution at room temperature for 2 h). Protein bands were visualised using enhanced chemiluminescence (ECL) (Amersham).

For immunofluorescence (Zhang et al, 2003), cells were grown on glass coverslips in 35 mm culture dishes, fixed for 20 min in 4% (v/v) formaldehyde and permeabilized by incubation for 5 min in 0.5% (v/v) Triton-X100 in PBS on ice. Subsequently, the cells were washed three times (10 min each) with PBS and incubated for 1 h at room temperature with a 1:100 dilution of rabbit polyclonal anti-TRPL antibody in PBS containing 5 m g/ml BSA as the blocking agent. After incubation for 1 h, cells were washed three times (10 min each) with PBS, then incubated with 1:500 goat anti-rabbit-IgG labelled with Cy3 in PBS at room temperature for 1 h. The cells were washed three times (10 min each) with PBS, and the coverslips mounted on a microscope slide in glycerol:PBS (80:20). Cy3 fluorescence was examined using an Olympus BX50 fluorescence microscope and 515-550 nM (excitation) and 575-615 (emission) filters. A video camera was used for image acquisition and digital images were saved as TIFF files.

For determination of the number of dead and living cells using the Trypan blue exclusion test, cells were grown in 35 mm culture dishes, the culture medium removed, and 1 ml of 0.1% (w/v) Trypan blue in PBS buffer added. The cells were incubated for 15-20 min at room temperature, the adhering cells viewed using a phase-contrast microscope (Nikon) equipped with an ocular square grid micrometer, and the number of Trypan blue-stained cells and the total number of cells per square millimeter determined for 5 different regions of the culture dish.

For estimation of the number of viable cells using 3-[4.5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) (Park et al, 1987), cells were grown at 37° C in a 96 well-plate in 200 m l of culture medium per well. MTT (20 m l of 5 mg/ml water) was added to each well, incubation was continued for a further 4 h, then 100 m l of 20% (w/v) SDS in 0.02 M HCl was added, the plates covered with aluminium foil, and incubated at room temperature overnight. The optical density of each well was measured at 570 nm (MTT) and 630 nm (background) using a microplate reader (Bio-Rad). Optical density readings were converted to readings of cell numbers using a calibration curve.

Chromatin DNA condensation and fragmentation in adherent and detached cells were assessed using Hoechst 33258 and by examination of nuclear morphology (Ferguson and Anderson, 1981; Yanagihara and Tsumuraya, 1992). Cells were grown in 35 mm culture dishes under the conditions described in the legends to figures. Detached cells from each dish were harvested by centrifugation (3,200xg at room temperature for 1 min), suspended in 0.5 ml of culture medium, mixed with 5 m l Hoechst 33258 (1 mg/ml in double-distilled water) and incubated for 10 min. A sample (15 m l) was transferred onto a glass slide. For analysis of adherent cells, the medium in the culture dishes containing the adherent cells was replaced with 1 ml culture medium, then 10 m l of Hoechst 33258 (1 mg/ml in double-distilled water) was added and the cells were incubated at room temperature. After 10 min, the cells were examined by fluorescence microscopy and the number of cells exhibiting condensed or fragmented nuclei as well as the total number of cells were determined.

Cells were grown on glass coverslips in 35 mm culture dishes were washed once with 1 ml complete RPMI-1640 medium at 37° C then incubated in 1 ml of Fura-2/AM loading solution, which was composed of 1 m l Fura-2/AM (3 m M in DMSO), 6.6 m l pluronic acid F-127 (10% w/v in DMSO (final concentration 0.02% v/v)) and 3.3 ml RPMI-5% FBS, for 30 min (at 37° C in 5% CO₂:95% O₂). After 30 min, the cells were washed three times with RPMI-5% FBS (37° C), then the coverslip placed in a coverslip holder and washed three times at 37° C with Ca²⁺-free modified Hank's solution. Modified Hank's solution (300 m l) was added and the coverslip holder placed in the microscope incubation chamber. The measurement of fura-2 fluorescence and conversion of fluorescence ratios to Ca²⁺ concentrations were performed as described previously (Gregory and Barritt, 2003). The initial rate of Ca²⁺ inflow was determined by calculating the slope of the plot of fluorescence ratio as function of time for the period 0-2 min after extracellular Ca²⁺ (2 mM) addition. The sustained rate of Ca²⁺ inflow was determined by calculating the slope of the plot of fluorescence ratio as function of time at 5, 10, 15 and 20 min after added Ca²⁺.

Unless otherwise indicated, results are expressed as mean ± S.E.M. with the number of experiments indicated in parentheses. Degree of significance were determined using Student's t-test for unpaired samples. Values of P < 0.05 were considered to be significant.

The TRPL protein and the luciferase reporter protein were transiently-expressed in PC-3 cells under the control of the Tet-On promoter by transfecting PT-20-1 cells (PC-3 cells stably-transfected with the Tet-On regulator) with the pBI-L-TRPL plasmid (which contains cDNA encoding the luciferase and TRPL proteins under the control of a bi-directional promoter). PT-20-1 cells transfected with the pBI-L plasmid (which does not contain cDNA encoding TRPL) were used as controls. Western blot analysis indicated that PT-20-1 cells transiently-transfected with pBI-L-TRPL exhibited a very low level of TRPL expression in the absence of doxycycline, and a doxycycline-inducible expression of the TRPL protein (Figure 1). Densitometric analysis indicated a 12-fold increase in TRPL protein expression (mean of two determinations) in cells grown in the presence of doxycycline. The doxycycline-induced increase in luciferase, which is a measure of transfection efficiency (Storz et al, 1999), was 6.6 ± 1.1 (n = 3) fold.

PT-20-1 cells transiently-transfected with pBI-L-TRPL exhibited a substantial doxycycline-induced increase in the number of dead cells, assessed using Trypan blue (Figure 2A), and a substantial increase in the number of cells exhibiting chromosome damage as assessed using Hoechst 33258 (Figure 2B). No doxycycline-induced changes in these parameters were observed in PT-20-1 cells transfected with the pBI-L or in untransfected PT-20-1 cells (Figure 2). In these experiments, the induction of luciferase by doxycycline was 7.2 ± 0.6 and 15.5 ± 1.9 (means ± SEM, n = 4) for cells transfected with pBI-L-TRPL and pBI-L, respectively.

The nuclear morphology of PT-20-1 cells transiently-transfected with the pBI-L or pBI-L-TRPL plasmids and stained with Hoechst 33258 (a fluorescent chromatin-binding dye (Ferguson and Anderson, 1981)) is shown in Figure 3. Both adherent and detached pBI-L-TRPL-transfected cells incubated in the absence of doxycycline exhibited some nuclear condensation (Figure 3E and 3G). The proportion of adherent and detached cells exhibiting nuclear condensation was increased when the pBI-L-TRPL-transfected cells were incubated in the presence of doxycycline (Figure 3F c.f. 3E and Figure 3H c.f. 3G). Cells transfected with pBI-L exhibited little nuclear condensation, and no increase in nuclear condensation when incubated in the presence of doxycycline (Figure 3A-3D). No doxycycline-induced increase in nuclear condensation was observed in PT-20-1 (non-transfected) cells (results not shown). The photomicrographs at higher magnification (Figure 3I-3L) demonstrate a variety of

Figure 1. Western blot analysis of the TRPL protein in extracts of Tet-On PC-3 cells transiently expressing TRPL. PT-20-1 (Tet-On PC-3) cells were transiently-transfected with pBI-L-TRPL using FuGENE6 and incubated in the absence of doxycycline for 24 h, then for a further 48 h in the absence or presence of doxycycline (1 m g/ml). Cells were harvested, crude membrane preparations prepared, and Western blot analysis conducted as described in Materials and Methods. TRPL protein present in Drosophila head extract was employed as a positive control. The arrow (128 kDa) indicates the position of the TRPL protein. The results shown are those obtained in one of two experiments which each gave similar results.

apoptotic nuclear morphologies in adherent PT-20-1 cells transiently-transfected with pBI-L-TRPL. There were many degraded nuclear forms (apoptotic nuclei) such as chromatin condensation with leakage into the cytoplasmic space (Figure 3I and 3J), DNA droplets of different size scattered throughout the cytoplasmic space (Figure 3L), localised chromatin condensation (Figure 3K), and general chromatin condensation (Figure 3J).

The numbers of condensed or fragmented nuclei in both adherent and detached cells were combined to estimate the total number of apoptotic nuclei. This was expressed as a percentage of all nuclei. For PT-20-1 cells transiently-transfected with pBI-L-TRPL grown in the presence of doxycycline the proportion of cells exhibiting condensed or fragmented nuclei was 19.5 ± 6.0 compared with 7.3 ± 3.0 for cells grown in the absence of doxycycline (mean ± SEM, n = 4), (P ¾ 0.05). There were no significant differences for either untransfected PT-20-1 cells or PT-20-1 cells transfected with pBI-L grown in the presence and absence of doxycycline (results not shown).

The results described in Figures 1-3 show that transient expression of the TRPL protein in PC-3 cells can be controlled by the Tet-On promoter system. The next experiments were performed to see if the same control of TRPL protein expression could be achieved in PC-3 cells stably expressing TRPL. PT-20-1 Tet-On PC-3 cells were stably-transfected with the pBI-L-TRPL plasmid as described in Materials and Methods. Two clones, MP25 and MP40, which showed the highest induction of the luciferase reporter gene were chosen for further study. Tests were made for the presence of the TRPL and luciferase genes in the cells grown over a number of passages. The resulting PCR products indicated that both the Trpl and luciferase genes were permanently incorporated into the cells (results not shown). When compared with the parent PT-20-1 Tet-On PC-3 cells, which were more spindle-shaped, cells of the MP40 and MP25 clones grown in the absence of doxycycline exhibited a homogenous rounded morphology. When grown in the presence of doxycycline, larger cells with a rounder shape were observed. These features of the cell morphology were observed in MP25 and MP40 cells grown over a number of passages.

When subjected to Western blot analysis, extracts of MP25 and MP40 cells grown in the absence of doxycycline exhibited a band at 128 kDa which corresponded in apparent size to the band of TRPL protein observed in Drosophila head extracts. When the cells were grown in the presence of doxycycline, the intensity of the 128 kDa band increased 1.3- and 1.4-fold (mean of two determinations) for MP25 and MP40 cells, respectively (results not shown). No band of 128 kDa was detected in extracts of PT-20-1 cells (Tet-On PC-3 cells which do not express TRPL) (results not shown).

Immunofluorescence staining of the TRPL protein was carried out to further investigate TRPL expression and its intracellular localisation. There was no difference in immunofluorescence in PT-20-1 cells grown in the absence or presence of doxycycline (Figure 4B c.f. 4A). By contrast, in the presence of doxycycline, MP40 cells exhibited increased fluorescence (Figure 4D c.f. 4C). Examination of the immunostained cells also confirmed the polymorphism and presence of a number of larger cells in MP40 cells grown in doxycycline (Figure 4D c.f. 4C).Similar results were obtained for MP25 cells (not shown). When MP25 and MP40 cells grown in the presence of doxycycline were examined at higher magnification the highest immunofluorescence intensity was observed around the perinuclear zone (Figure 4E-4H) indicating the presence of considerable amounts of TRPL protein in this region.

Figure 2. Transient expression of TRPL in Tet-On PC-3 cells leads to a doxycycline-inducible increase in cell death (assessed using Trypan blue) (A) and an increase in the number of cells exhibiting chromosome damage, chromatin condensation, and chromatin fragmentation (assessed using Hoechst 33258) (B). PT-20-1 (Tet-On PC-3) cells were transiently-transfected with the pBI-L (control) or pBI-L-TRPL plasmids (1 m g) using Lipofectamine. After 4 h, the medium was changed and the cells grown in the absence and presence of doxycycline (1 m g/ml) for 48 h. The number of cells excluding Trypan blue and the number exhibiting chromatin condensation or fragmentation were determined as described in Materials and Methods. In each case, the results are expressed as a percentage of the total number of cells present under the given condition tested. The results are the means ± S.E.M. (n = 4)

Figure 3. Patterns of nuclear chromatin condensation and fragmentation in Tet-On PC-3 cells transiently expressing TRPL. PT-20-1 (Tet-On PC-3) cells were transiently-transfected with pBI-L (control) (A-D) or pBI-L-TRPL (E-H and I-L) and incubated in the absence or presence of doxycycline (1 m g/ml) for 48 h, then stained with Hoechst 33258. Magnification: x200 (A-H) and x400 (I-L). Cell transfection and culture, the harvesting of detached cells, the staining of adherent and detached cells with Hoechst 33258, and fluorescence microscopy were performed as described in Materials and Methods. The images shown are those obtained from one of 5 or more experiments which each gave similar results.

Figure 4. Immunofluorescence images of the TRPL protein expressed in two stable Tet-On PC—3 clones (MP25 and MP40) expressing TRPL. A-D. PT-20-1 (Tet-On PC-3) cells (A,B) and MP40 cells (Tet-On PC-3 cells stably expressing TRPL) (C,D) cultured for 48 h in the absence (A,C) or presence (B,D) of doxycyline (4 m g/ml). Magnification: x200. E-H. MP25 (Tet-On PC-3 cells expressing TRPL) (E,F) and MP40 (G,H) cells cultured for 48 h in the presence of doxycycline (4 m g/ml). Magnification: x400. Cell culture and the detection of TRPL by immunofluorescence were performed as described in Materials and Methods. The results shown are representative of those obtained for more than 20 fields of cells which showed similar morphology.

Basal values of [Ca²⁺]_cyt were measured in cells loaded with fura-2 and incubated in the absence of added extracellular Ca²⁺. Initial and sustained rates of Ca²⁺ inflow were measured following the addition of 2 mM extracellular Ca²⁺. When grown in either the presence or absence of doxycycline, PC-20-1 cells (control, no TRPL expression) exhibited no difference in basal [Ca²⁺]_cyt or in the initial and sustained rates of Ca²⁺ inflow (Figure 5A and 5D-5F).

When compared with cells grown in the absence of doxycycline, MP25 and MP40 cells (expressing TRPL) grown in the presence of doxycycline exhibited an increased value of basal [Ca²⁺]_cyt and greater initial and sustained rates of Ca²⁺ inflow (Figure 5B-5F).

The effect of TRPL expression on cell viability was assessed using the MTT test. As described above, when grown in the presence of doxycycline, MP40 cell cultures exhibited numerous swollen large cells (Figure 6A).

Figure 5. Stable expression of TRPL in Tet-On PC-3 cells leads to a doxycycline-inducible enhancement of the sustained rate of Ca²⁺ inflow. A-C. Plots of fluorescence as a function of time for PT-20-1 (Tet-On PC-3) cells (A) and MP25 (B) and MP40 cells (C) (Tet-On cells stably expressing TRPL). Cells were grown for 48 h in the presence or absence of doxycycline (4 m g/ml), loaded with fura-2, and fluorescence measured as a function of time, as described in Materials and Methods. Cells were initially incubated in Ca²⁺-free Hank's medium in the absence of added extracellular Ca²⁺. At the time indicated by the arrows, CaCl₂ (2 mM) was added to the incubation medium. The traces shown are representative of those obtained in three experiments which each gave similar results. D-F. Basal values of [Ca²⁺]_cyt (D) and initial (E) and sustained (F) rates of Ca²⁺ inflow for PT-20-1, MP25 and MP40 cells. Values of [Ca²⁺]_cyt, and initial and sustained rates of Ca²⁺ inflow were determined as described in Materials and Methods. The results are the means ± S.E.M. (n = 3).

Figure 6. Tet-On PC-3 cells stably expressing TRPL exhibit a doxycycline-inducible decrease in cell proliferation (assessed using the MTT assay) when grown in culture. A. A photomicrograph of MP40 cells grown for 48 h in the presence of doxycycline (4 m g/ml). Magnification: x200. B-D. The number of viable cells plotted as a function of time for PT-20-1 (Tet-On PC-3) cells (B), MP25 (C) and MP40 (D) cells (Tet-On PC-3 cells stably expressing TRPL) grown in the presence or absence of doxycycline (4 m g/ml). PT-20-1 cells (B), and MP25 (C) and MP40 (D) cells were plated in 96 well-plates for 24 h, then grown in the absence or presence of doxycycline (4 m g/ml) and the number of viable cells determined using the MTT assay, as described in Materials and Methods. The results are the means ± S.E.M. (n = 4). The degree of significance (*), determined using Student's t-test for unpaired samples, is P ¾ 0.05.

These large cells disintegrated easily, and detached from the surface of the culture dish. For PT-20-1 cells (control, no TRPL expression), culture in the presence of doxycycline did not cause any difference in the number of viable cells when assessed using the MTT test (Figure 6B). By contrast, growth of MP25 and MP40 cells in the presence of doxycycline reduced the number of viable cells (Figure 6C and 6D). The number of apoptotic nuclei was assessed by determining the level of chromatin condensation and nuclear fragmentation using Hoechst 33258. Representative photomicrographs of PT-20-1 and MP40 cell cultures stained with Hoechst 33258 are shown in Figure 7. For PT-20-1 cells grown in either the absence or presence of doxycycline, the number of adherent cells exhibiting chromatin condensation and fragmentation was small (Figure 7B c.f. 7A, and Figure 7H). Moreover, no detached ("floating") cells were observed. For MP25 and MP40 cultures grown in the absence of doxycycline, the number of adherent cells exhibiting chromatin condensation and fragmentation at a given time was slightly greater compared to the number observed for PT-20-1 cultures grown in the absence of doxycycline (Figure 7C and Figure 7H). In the presence of doxycycline, the number of adherent cells exhibiting chromatin condensation and fragmentation at a given time after doxycycline addition was increased (Figure 7D and Figure 7H). Chromatin condensation was observed more frequently than fragmented nuclei. Examples are shown in Figure 7E-7G. The variability in the percentage of adherent cells exhibiting chromatin condensation and nuclear fragmentation (Figure 7H) is likely to be due to the disintegration of swollen cells (c.f. the cells shown in the photomicrography in Figure 6A).

The key observations reported here are that (i) expression of the TRPL protein in PC-3 prostate cancer cells can be controlled by the tetracycline-responsive Tet-On promoter system, (ii) the Tet-On system is effective when TRPL (under the control of the TRE element) is expressed either transiently or stably, and (iii) tetracycline-

Figure 7. Tet-On PC-3 cells stably expressing TRPL exhibit a doxycycline-inducible increase in the number of cells with nuclear chromatin condensation and fragmentation (assessed using Hoechst 33258) when grown in culture. A-D. PT-20-1 (Tet-On PC-3) cells (A,B) and MP40 cells (Tet-On PC-3 cells stably expressing TRPL) (C,D) grown for 48 h in the presence (B,D) and absence (A,C) of doxycycline (4 m g/ml) then stained with Hoechst 33258. Magnification: x200. E-J. Adherent (E-G) MP40 cells grown for 48 h in the presence of doxycycline (4 m g/ml) then stained with Hoechst 33258. Magnification: x400. Cell culture, the harvesting of detached cells, staining with Hoechst 33258, and fluorescence microscopy were performed as described in Materials and Methods. The images shown are those obtained from one of 10 or more experiments which each gave similar results. H. Cells grown as described above for 48, 72 or 96 h. At each time point, the cells adherent to the culture dish were stained with Hoechst 33258, examined by fluorescence microscopy, and the number of cells exhibiting fragmented or condensed nuclei was determined and expressed as a percentage of the total number of cells present at that time point. The results are the means ± S.E.M (n = 3).

induced expression of TRPL is associated with cell toxicity and death. The results provide an "in principle" demonstration that controlled expression of: the TRPL protein can kill prostate cancer cells. This inducible system could be further refined and developed by optimising TRPL expression and targetting expression of the TRPL protein and the inducible promoter to prostate cancer cells. The inducible TRPL Ca²⁺ channel expression system offers a potential therapeutic strategy for the killing of androgen-independent prostate cancer cells. It could also be applied to any other cancer cell type in which the TRPL protein can be over-expressed.

The transient transfection of Tet-On PC-3 cells with TRPL DNA under control of the TRE element provided a clear and significant doxycycline induction of expression

of the TRPL (and luciferase reporter gene) proteins, decreased cell viability and increased apoptosis. The following were also clearly evident in Tet-On PC3 cells stably transfected with TRPL DNA under control of the TRE element. Doxycycline-induced expression of the TRPL and the luciferase proteins, increased basal [Ca²⁺]_cyt, changes in cell morphology, and increased cell death and apoptosis. However, the degree of induction of both the TRPL and luciferase proteins was lower in stably-transfected cells than that observed in transiently-transfected cells. Also, it proved quite difficult to isolate stable Tet-On clones expressing TRPL. This may have been due to the presence in PC-3 cells of proteins other than the Tet-On transcription factor which can activate the TRE element in the absence of doxycycline. This, in turn, would induce TRPL expression in the absence of doxycycline and cause subsequent Ca²⁺ and Na⁺ inflow and some cell death.

While TRPL was successfully expressed in PC-3 cells under the control of the Tet-On inducible promoter system, there was only a small response to doxycycline in the two stable double-transfected clones (MP25 and MP40) which were studied in detail. Moreover, the stable double-transfected clones exhibited a relatively high basal luciferase expression and only a modest doxycycline induction of luciferase which was lost in later passages. By contrast, when either PT-20-1 cells (Tet-On PC-3 cells expressing the Tet-On regulator (rTetR/VP16)) or stable double-transfected cells were transiently-transfected with the pTRE-Luc plasmid (harbouring DNA encoding the TRE-response element and luciferase), doxycycline induced a substantial increase in luciferase expression. This demonstrates the effectiveness of the Tet-On regulatory system in PC-3 cells and indicates that the induction machinery (expression of rTetR/VP16 and formation of the rTetRVP16-doxycycline complex) is intact. The degree of doxycycline-induced luciferase expression in cells transiently expressing pTRE-Luc was comparable with that observed by others for the Tet-On system in stable cell lines (Gschwend et al, 1997).

While the induction by doxycycline of luciferase and TRPL in double-stable transfected PC-3 cells was small, it is within the wide range reported for the expression of proteins under the control of the TRE promoter element in other cell types (Nakamura et al, 2000; Gossen and Bujard, 1992). In contrast to the results reported here for luciferase expression in PC-3 cells, others have reported the successful stable expression of luciferase in PC-3 cells under the control of the CMV promoter (Rubio et al, 1998, 2000) and the inducible TRE promoter element in the Tet-off PC-3 cell line (Gschwend et al, 1997). However, the plasmids employed in these other studies (Gschwend et al, 1997; Rubio et al, 1998, 2000) differ from those employed here. High background levels (leakage) of proteins expressed under control of the TRE promoter element have also been reported for the Tet-Off system in PC-3 cells (Gschwend et al, 1997) and for the TRE system in other cell types (Park and RajBhandary, 1998; Keyvani et al, 1999).

The high basal levels of luciferase and TRPL expression observed in the absence of doxycycline in double-stable transfected PC-3 clones (MP25 and MP40) may be due to the presence of endogenous proteins which activate the TRE promoter element and/or the minimal CMV promoters (Gschwend et al, 1997; Park and RajBhandary, 1998; Keyvani et al, 1999), a high basal activity of the minimal CMV promoter in the PC-3 cell environment (Gossen and Bujard, 1992, 1995; Gossen et al, 1993), incomplete integration of the pBI-L-TRPL plasmid (Gossen and Bujard, 1992), and/or high plasmid copy number (Gill and Ptashne, 1988). However, it is noted that such high basal levels of expression were not observed in transiently-transfected PT-20-1 Tet-On PC-3 cells. Reasons for the lack of substantial induction by doxycycline of luciferase and TRPL expression may also include the possibility that expressed luciferase accumulates in peroxysomes and cannot be readily solubilized under the conditions of the luciferase assay (Keller et al, 1987), toxic effects of the expressed TRPL protein which affect expression of both luciferase and TRPL (Garcia-Gallo et al, 1999; Liu et al, 1999), and location of the minimal CMV promoter of the pBI-L-TRPL plasmid in a region where it cannot be activated by the doxycycline-rTetR-VP16 complex (Gossen and Bujard, 1995; Freundlieb et al, 1999).

The process by which TRPL expression kills PC-3 cells is most likely initiated by enhanced Ca²⁺ and Na⁺ inflow across the plasma membrane. The present results show that the toxic effects of expression of TRPL are associated with increased Ca²⁺ inflow and elevated [Ca²⁺]_cyt. Since TRPL is a non-selective Ca²⁺ channel which admits Na⁺ as well as Ca²⁺ (Kunze et al, 1997), it is likely that an increased intracellular Na⁺ concentration accompanies the observed increase in [Ca²⁺]_cyt. The idea that Na⁺ plays a role in cell toxicity is consistent with the observation that cell swelling followed by cell disintegration and chromosome degradation are not typical of cells undergoing apoptosis or necrosis (Kroemer et al, 1998; Hacker, 2000). This suggests that, at the very least, Na⁺ as well as Ca²⁺ is involved in the pathway of cell toxicity. The morphology of PC-3 cells stably expressing TRPL (large, polymorphic, and swollen cells) is similar to that of HepG2 and MKN42-P cells heterologously expressing the MDEG channel which induced a massive Na⁺ inflow (Horimoto et al, 2000). The TRPL-mediated increase in [Ca²⁺]_cyt may cause other changes, including disruption of mitochondria (Beaver and Waring, 1994; McConkey and Orrenius, 1997; Berridge et al, 1998; Crompton, 1999; Jambrina et al, 2003), activation of Ca²⁺/Mg²⁺-dependent endonucleases (Cohen and Duke, 1984; Ribeiro and Carson, 1993) and K⁺ efflux (Speake et al, 1998) which, in part, lead to apoptosis. It is interesting to note that expression of Bcl-2 and Bcl-xL has been reported to decrease [Ca²⁺]_cyt (McDonnell et al, 1992; Porn-Ares et al, 1998; Lebedeva et al, 2000; Scorrano et al, 2003). The presence of these proteins in PC-3 cells may counteract the effects of increased [Ca²⁺]_cyt due to TRPL expression.

Another possibility is that cation flow across intracellular membranes and/or the physical disruption of these membranes by large amounts of intracellular TRPL are partly responsible for some of the toxic effects of TRPL. A considerable amount of the stably-expressed TRPL protein appeared to be located at intracellular sites, especially around the nucleus. These sites may be the Golgi and endoplasmic reticulum. Such an intracellular location of several heterologously-expressed TRP proteins has previously been observed by others (Wang et al, 1999; Schaefer et al, 2000; Hofmann et al, 2002). The location of TRPL at intracellular sites may represent the accumulation of large amounts of TRPL in vesicles which are part of the trafficking pathway to the plasma membrane (Wang et al, 1999; Schaefer et al, 2000; Hofmann et al, 2002) or may be part of a normal mechanism for the activation of members of the TRP channel family by exocytosis (e.g. Bezzerides et al, 2004).

In conclusion, the results of the present study demonstrate, in principle, that TRPL expression in prostate cancer cells can be controlled by an inducible promoter system. The mechanism by which the expressed TRPL protein induces cell death is likely to involve increased intracellular Ca²⁺ and Na⁺, changes in the morphology and biochemistry of intracellular organelles, and the apoptotic pathway. This system offers a potential strategy for the controlled killing of prostate and other cancer cells.

The authors gratefully acknowledge Dr. Len Kelly (Department of Genetics, University of Melbourne) for provision of TRPL cDNA, the advice of Dr. Tony Edwards, School of Medicine, Flinders University, and Ms. Courtney Bryans and Mrs. Diana Kassos for preparing the typescript. Support by a grant from the Cancer Council South Australia and the Thai Government for a postgraduate research scholarship are gratefully acknowledged.