I. Introduction

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 inconcentration 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.

II. Materials and methods

A. Materials

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-MEM^â 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).

B. Cell culture

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 mg 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 mg/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 mg/ml G418 and 25 mg/ml hygromycin.

C. Plasmids

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 mF, 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.

D. Transfection protocols

Electroporation of PC-3 cells (Bio-Rad Gene Pulser) was performed at 500 volts and 25 mF. 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 ml) and RPMI-1640 (serum free) (97 ml) 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 mg in 200 ml Opti-MEM^â)with lipofectamine (3 ml in 200 ml Opti-MEM^â) 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.

E. Protocol for the isolation and storage of stably-transfected cell lines

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 cm²culture 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.

F. Generation of stably-transfected clones of PC-3 cells harbouring the Tet-On plasmid

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 mg/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 mg/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.

G. Generation of stable clones of Tet-On PC-3 cells harbouring cDNA encoding the TRPL and luciferase genes

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 mg/ml) and G418 (100 mg/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 mg/ml) (in order to increase the selection pressure) while maintaining the concentration of G418 at 100 mg/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 mg/ml hygromycin and G418 (100 mg/ml) for a further two passages, then maintained in the presence of a low concentration of hygromycin (25 mg/ml) plus G418 (100 mg/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 mg/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.

H. PCR and luciferase assay

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 mM of each dNTP, 0.4 mM of each primer (sense, antisense), and 0.4 units Taq polymerase in 1x Taq PCR buffer. Each reaction mixture (23 ml) was overlaid with mineral oil then 2 ml 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 ml 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 ml 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.

I. Western blot analysis and immunofluorescence

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 ml 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 10⁶cells), 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 ml 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 10⁶cells), 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 ml 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 mg/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.

J. Determination of cell viability and chromatin condensation and fragmentation

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 ml of culture medium per well. MTT (20 ml of 5 mg/ml water) was added to each well, incubation was continued for a further 4 h, then 100 ml 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 ml Hoechst 33258 (1 mg/ml in double-distilled water) and incubated for 10 min. A sample (15 ml) 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 ml 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.

K. Cytoplasmic Ca²⁺ concentration and initial and sustained rates of Ca²⁺ inflow

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 ml Fura-2/AM (3 mM in DMSO), 6.6 ml 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 ml) 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⁺.

L. Expression of results

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.

III. Results

A. Effects of transient expression of the TRPL protein in Tet-On PC-3 cells on cell viability and nuclear fragmentation

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

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.

B. Comparison of the transient and stable transfection of PC-3 cells with TRPL DNA under control of the TRE element

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).

C. Possible mechanisms of TRPL-induced cell death

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⁺]_cytdue 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.

Acknowledgements

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.

References

Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M. Cerwinski W, MacDonald JF, Tymianski M (2003) A key role for TRPM7 channels in anoxic neuronal death. Cell 115, 863-877

Beaver JP, Waring P (1994) Lack of correlation between early intracellular calcium ion rises and the onset of apoptosis. Immunol Cell Biol. 72, 489-499

Berridge MJ, Bootman MD, Lipp P (1998) Calcium – a life and death signal. Nature. 395, 645-648

Bezzerides VJ, Ramsey IS, Kotecha S, Greka A, Clapham DE (2004) Rapid vesicular translocation and insertion of TRP channels. Nature Cell Biology 6, 709-720

Bradford HF, Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Adv Exp Med Biol. 69, 395-404

Cohen JJ, Duke RC (1984) Glucocortcoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J Immunol. 132, 38-42

Colombel MG, Gil Diez S, Radvanyi F, Buttyan R, Thiery JP, Chopin D (1996) Apoptosis in prostate cancer. Molecular basis of study hormone refractory mechanisms. In: Castagnetta INL, Leon Bradlow H (eds). Basis for Cancer Management. The New York Academy of Sciences, New York, New York

Crompton M (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem J. 341, 233-249

Denmeade SR, Isaacs JT (1996) Activation of programmed (apoptotic) cell death for the treatment of prostate cancer. Adv Pharmacol. 35, 281-306

Domann R, Martinez J (1995) Alternative to cloning cylinders for isolation of adherent cell clones. Biotechniques. 18, 594-595

Ferguson DJ, Anderson TJ (1981) Ultrastructural observations on cell death by apoptosis in the “resting” human breast. Virchows Arch A Pathol Anat Histol. 393, 193-203

Freundlieb S, Schirra-Muller C, Bujard H (1999) A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells. J Gene Med. 1, 4-12

Furuya Y, Lundmo P, Short AD, Gill DL, Isaacs TJ (1994) The role of calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cells induced by thapsigargin. Cancer Res. 54, 6167-6175

Garcia-Gallo M, Behrens MM, Renart J, Diaz-Guerra M (1999) Expression of N-methyl-D-asparate receptors using vaccinia virus causes exocytotic death in human kidney cells. J Cell Biochem. 72, 135-144

Gill G, Ptashne M (1988) Negative effect of the transcriptional activator GAL4. Nature. 334, 721-724

Gossen M, Bonin AL, Bujard H (1993) Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements. Trends Biochem Sci. 18, 471-475

Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA. 89, 5547-5551

Gossen M, Bujard H (1995) Efficacy of tetracycline-controlled gene expression is influenced by cell type: commentary. Biotechniques. 19, 213-217

Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H (1995) Transcriptional activation by tetracyclines in mammalian cells. Science. 268, 1766-1769

Gregory RB, Barritt GJ (2003) Evidence that Ca²⁺-release-activated channels in rat hepatocytes are required for the maintenance of hormone-induced Ca²⁺ oscillations. Biochem J. 370, 695-702

Gschwend JE (1996) Apoptosis: principles and importance of programmed cell death for prostatic carcinoma. Urologe A. 35, 390-399

Gschwend JE, Fair WR, Powell CT (1997) Evaluation of the tetracycline-repressible transactivator system for inducible gene expression in human prostate cancel cell lines. Prostate. 33, 166-176

Gutierrez AA, Arias JM, Garcia L, Mas-Oliva J, Guerrero-Hernandez A (1999) Activation of a Ca²⁺-permeable cation channel by two different inducers of apoptosis in a human prostatic cancer cell line. J Physiol. 517, 95-107

Hacker G (2000) The morphology of apoptosis. Cell Tissue Res. 301, 5-17

Hofmann T, Schaefer M, Schultz G, Gudermann T (2002) Subunit composition of mammalian transient receptor potential channels in living cells. Proc Natl Acad Sci USA. 99, 7461-7466

Horimoto M, Sasaki Y, Ugawa S, Wada S, Toyama T, Iyoda K, Yakushijin K, Minami Y, Ito T, Hijioka T, Eguchi A, Nakanishi M, Shimada S, Tohyama M, Hayashi N, Hori M (2000) A novel strategy for cancer therapy by mutated mammalian degenerin gene transfer. Cancer Gene Therapy. 7, 1341-1347

Isaacs JT, Lundmo PI, Berges R, Martikainen P, Kyprianou N, English HF (1992) Androgen regulation of programmed death of normal and malignant prostatic cells. J Androl. 13, 457-464

Jambrina E, Alonso R, Alcalde M, del Carmen Rodr£guez M, Serrano A, Mart£nez-A C, Garc£a-Sancho J, Izquierdo M (2003) Calcium influx through receptor-operated channels induces mitochondria-triggered paraptotic cell death. J Biol Chem. 278, 14134-14145.

Kass GE, Orrenius S (1999) Calcium signalling and cytotoxicity. Environ Health Perspect. 107 Suppl 1, 25-35

Keller GA, Gould S, Deluca M, Subramani S (1987) Firefly luciferase is targeted to peroxisomes in mammalian cells. Proc Natl Acad Sci USA. 84, 3264-3268

Keyvani K, Baur I, Paulus W (1999) Tetracycline-controlled expression but not toxicity of an attenuated dipthelia toxin mutant. Life Sci. 64, 1719-1724

Kroemer G, Dallaporta B, Resche-Rigon M (1998) The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol. 60, 619-642

Kunze DL, Sinkins WG, Vaca L, Schilling WP (1997) Properties of single Drosophila Trpl channels expressed in Sf9 insect cells. Am J Physiol. 272, C27-C34

Lebedeva I, Rando R, Ojwang J, Cossum P, Stein CA (2000) Bcl-xL in prostate cancer cells: effects of overexpression and down-regulation on chemosensitivity. Cancer Res. 60, 6052-6060

Liu HS, Jan MS, Chou CK, Chen PH, Ke NJ (1999) Is green fluorescent protein toxic to the living cells? Biochem Biophys Res Commun. 260, 712-717

Martikainen P, Kyprianou N, Tucker RW, Isaacs JT (1991) Programmed death of nonproliferating androgen-independent prostatic cancer cells. Cancer Res. 51, 4693-4700

McConkey DJ, Orrenius S (1997) The role of calcium in the regulation of apoptosis. Biochem Biophys Res Commun. 239, 357-366

McDonnell TJ, Troncoso P, Brisbay SM, Logothetis C, Chung LW, Hsieh JT, Tu SM, Campbell ML (1992) Expression of the protooncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer. Cancer Res. 52, 6940-6944

Nakamura K, Bossy-Wetzel E, Burns K, Fadel MP, Lozyk M, Goping IS, Opas M, Bleackley RC, Green DR, Michalak M (2000) Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis. J Cell Biol. 150, 731-740

Niemeyer BA, Suzuki E, Scott K, Jalink K, Zuker CS (1996) The Drosophila light-activated conductance is composed of the two channels TRP and TRPL. Cell. 85, 651-659

Park H-J, RajBhandary UL (1998) Tetracycline-regulated suppression of amber codons in mammalian cells. Mol Cell Biol. 18, 4418-4425

Park JG, Kramer BS, Steinberg SM, Carmichael J, Collins JM, Minna JD, Gazdar AF (1987) Chemosensitivity testing of human colorectal carcinoma cell lines using a tetrazolium-based colorimetric assay. Cancer Res. 47, 5875-5879

Phillips AM, Bull A, Kelly LE (1992) Identification of a Drosophila gene encoding a calmodulin-binding protein with homology to the trp phototransduction gene. Neuron. 8, 631-642

Porn-Ares MI, Ares MPS, Orrenius S (1998) Calcium signalling and the regulation of apoptosis. Toxicol In Vitro. 12, 539-543

Putney Jr JW, Broad LM, Braun FJ, Lievremont JP, Bird GS (2001) Mechanisms of capacitative calcium entry. J Cell Sci. 114, 2223-2229

Ribeiro JM, Carson DA (1993) Ca²⁺/Mg²⁺-dependent endonuclease from human spleen: purification, properties, and role in apoptosis. Biochem. 32, 9129-9136

Rubio N, Villacampa MM, Blanco J (1998) Traffic to lymph nodes of PC-3 prostate tumor cells in nude mice visualised using the luciferase gene as a tumor cell marker. Lab Invest. 78, 1315-1325

Rubio N, Villacampa MM, El Hilali N, Blanco J (2000) Metastatic burden in nude mice organs measured using prostate tumor PC-3 cells expressing the luciferase gene as a quantifiable tumor cell marker. Prostate 44, 133-143

Schaefer M, Plant TD, Obukhov AG, Hofmann T, Gudermann T, Schultz G (2000) Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5. J Biol Chem. 275, 17517-17526

Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T, Korsmeyer SJ (2003) BAX and BAK regulation of endoplasmic reticulum Ca²⁺: a control point for apoptosis. Science. 300, 135-139

Skryma R, Mariot P, Bourhis XL, Coppenolle FV, Shuba Y, Vanden Abeele F, Legrand G, Humez S, Boilly B, Prevarskaya N (2000) Store depletion and store-operated Ca²⁺ current in human prostate cancer LNCaP cells: involvement in apoptosis. J Physiol. 527, 71-83

Speake T, Douglas IJ, Brown PD (1998) The role of calcium in the volume regulation of rat lacrimal acinar cells. J Membr Biol. 164, 283-291

Steinsapir J, Socci R, Reinach P (1991) Effects of androgen on intracellular calcium of LNCaP cells. Biochem Biophys Res Commun. 179, 90-96

Storz P, Doppler H, Horn-Muller J, Groner B, Pfizenmaier K, Muller G (1999) A cellular reported assay to monitor insulin receptor kinase activity based on STAT 5-dependent luciferase gene expression. Anal Biochem. 276, 97-104

Tilley WD, Bentel JM, Aspinall JO, Hall RE, Horsfall DJ (1995) Evidence for a novel mechanism of androgen resistance in the human prostate cancer cell line, PC-3. Steroids. 60, 180-186

Tombal B, Gailly P, Van Cangh PJ, Gillis JM (1995) Role of intracellular calcium in the programmed cell death of prostatic cancer cells. Acta Urol Belg. 63, 1-5

Wang W, O’Connell B, Dykeman R, Sakai T, Delporte C, Swaim W, Zhu X, Birnbaumer L, Ambudkar IS (1999) Cloning of Trp1ß isoform from rat brain: immunodetection and localization of the endogenous TRP1 protein. Am J Physiol. 276, C969-C979

Webber MM, Bello D, Quader S (1997) Immortalised and tumorigenic adult human prostatic epithelial cell lines: characteristics and applications Part 2. Tumorigenic cell lines. Prostate 30, 58-64

Yanagihara K, Tsumuraya M (1992) Transforming growth factor beta 1 induces apoptotic cell death in cultured human gastric carcinoma cells. Cancer Res. 52, 4042-4045

Zhang L, Brereton HM, Hahn M, Froscio M, Tilley WD, Brown MP, Barritt GJ (2003) Expression of the Drosophila Ca²⁺ permeable transient receptor potential-like (TRPL) channel protein in a prostate cancer cell line decreases cell survival. Cancer Gene Therapy. 10, 611-625

Zimmer S, Trost C, Wissenbach U, Philipp S, Freichel M, Flockerzi V, Cavalie A (2000) Modulation of recombinant transient-receptor-potential-like (TRPL) channels by cytosolic Ca²⁺. Pflugers Arch. 440, 409-417.

Gregory J. Barritt

Research Article

Received: 11 August 2004; Accepted: 18 October 2004; electronically published: October 2004