Skip Navigation


NDT Advance Access originally published online on January 18, 2006
Nephrology Dialysis Transplantation 2006 21(5):1278-1288; doi:10.1093/ndt/gfk036
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
21/5/1278    most recent
gfk036v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Wang, W.
Right arrow Articles by Dai, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, W.
Right arrow Articles by Dai, B.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Clinical Nephrology

Aberrant expression of SPARC and its impact on proliferation and apoptosis in ADPKD cyst-lining epithelia

Wenjing Wang, Changlin Mei, Bing Tang, Haidan Zhao, Chenggang Xu, Zhanyuan Li, Xuefei Shen, Wencheng Fu and Bing Dai

Division of Nephrology, Center of Kidney Disease, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai 200003, China

Correspondence and offprint requests to: Changlin Mei, Division of Nephrology, Center of Kidney Disease, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai 200003, China. Email: chlmei{at}public1.sta.net.cn



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Autosomal dominant polycystic kidney disease (ADPKD) results from a combination of environmental and genetic factors. Secreted protein acidic and rich in cysteine (SPARC) can be expressed by many different cell types and is associated with development, remodelling, cell turnover and tissue repair. The analysis of SPARC would help evaluate the effect of the unique matricellular glycoprotein on renal disease progression in ADPKD.

Methods. The concentration of SPARC was measured with an enzyme-linked immunosorbent assay (ELISA); distribution and expression levels were measured with in situ hybridization, immunohistochemistry, reverse transcription–polymerase chain reaction (RT–PCR) and western blot assays. Apoptosis was assessed by morphological observation and fluorescence-activated cell sorting (FACS) apoptosis index (AI) analysis. Cell cycle phase was examined by FACS analysis. Cell proliferation was studied using bromodeoxyuridine (BrdU) incorporation ELISA.

Results. The SPARC level in the renal cyst fluid of patients with ADPKD was greater than that in patients with simple renal cyst (SRC), and also greater than that found in the plasma and urine of patients with either ADPKD or SRC and normal subjects. SPARC mRNA and protein levels in polycystic renal tissue were greater than that in normal renal tissue. Additionally, SPARC could inhibit cyst-lining epithelial cell proliferation, bring about cell cycle arrest in the G0/G1 phase and induce apoptosis in vitro. SPARC treatment resulted in decreased mRNA levels of PCNA (proliferating cell nuclear antigen), MCM2 (minichromosome maintenance protein 2), ClnD1 and Bcl-2, but an increased mRNA level of p21Waf1 in cyst-lining epithelial cells.

Conclusion. Our findings suggest that the increased SPARC expression in ADPKD renal tissue may provide negative feedback in ADPKD patients.

Keywords: ADPKD; proliferation; SPARC



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common life-threatening monogenic hereditary diseases with an incidence rate of ~1:1000 afflicting 5–6 million people in the world [1]. The occurrence and development of cysts are due to a ‘two hit’ of the PKD gene, resulting in an abnormality of cell cycle regulation and intercellular metabolism. This dysregulation of the cell cycle and cellular metabolism subsequently results in epithelial hyperplasia and accumulation of liquid in the renal tubule, and finally leads to the development and gradual enlargement of bilateral renal cysts [2,3]. The abnormalities of the extracellular matrix, such as fibrin and laminin, also accelerate the development of ADPKD. About 50% of PKD patients develop end-stage renal failure (ESRF) by late middle age [4], accounting for 5–10% of patients requiring haemodialysis [1]. ADPKD can have effects on other organs, including polycystic liver, pancreatic and bile duct dilation, colonic diverticula, intracranial aneurysm and abnormality of the cardiac valve [5]. Despite the severity and incidence of ADPKD, there unfortunately remains a lack of effective therapeutic methods [6].

Secreted protein, acidic and rich in cysteine (SPARC), also termed osteonectin or BM-40, is a calcium-binding matricellular glycoprotein with antiproliferative and counteradhesive properties that can interact with specific cytokines and growth factors such as platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF), bind to structural proteins and interface with cells directly. It plays important roles in embryogenesis, tissue remodelling, wound repair, morphogenesis, cellular differentiation, cell migration and angiogenesis [7]. SPARC, together with thrombospondin 1, tenascin C, TSP2, tenascin X, syndecans and osteopontin, comprise a non-homologous functional group of secreted matricellular proteins that interact with cell surface receptors, the extracellular matrix, growth factors and/or proteases but do not in themselves subserve exclusively structural roles [8,9].

Recent findings indicate that SPARC is involved in glomerular remodelling and repair [10,11]. Furthermore, SPARC has been implicated in various renal diseases such as passive Heymann nephritis, mesangioproliferative glomerulonephritis and diabetic nephropathy [10,11]. However, little is known about the role of SPARC in the progression of ADPKD.

We previously found by cDNA expression microarray that SPARC is upregulated in ADPKD kidney tissues (unpublished observation). Thus, in this study, we proceeded to investigate the role of SPARC in ADPKD. Body fluid concentrations of SPARC were measured and the distribution of SPARC in kidney tissues of patients with ADPKD was observed. In addition, ADPKD cyst-lining epithelial cells were treated directly with exogenous SPARC and their growth and apoptosis were studied. The effects of SPARC treatment on the mRNA levels of proliferation-related genes [proliferating cell nuclear antigen (PCNA) and minichromosome maintenance protein 2 (MCM2)], cell cycle regulating genes (ClnD1 and p21Waf1) and apoptosis-related genes (Bax and Bcl-2) were assessed.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Plasma, urine and cyst fluid samples
Samples of plasma, urine and cyst fluid were obtained from 37 Han subjects (16 males, 21 females; aged 18–78 years, mean age 47.41±12.63 years) with ADPKD and 10 Han subjects with simple renal cyst (SRC) (four males, six females; aged 33–65 years, mean age 49.35±13.26 years old). Control samples of plasma and urine were obtained from 20 Han volunteers (nine males, 11 females; aged 35–70 years, mean age 51.75±14.07 years). All SRC subjects had unilateral renal cysts without hypertension, proteinuria or renal dysfunction. The subjects with ADPKD included out-patients and in-patients of Shanghai Changzheng Hospital. Chronic kidney disease (CKD) classification was evaluated by the Kidney Disease Outcomes Quality Initiative (K/DOQI) [12]. Glomerular filtration rate (GFR) was estimated using the MDRD prediction equation and given in ml/min/1.73 m2 [170 x (creatinine)–0.999 x (age)–0.76 x (urea)–0.170 x (albumin)+0.318 x 0.762 if female] based on age and levels of serum creatinine, urea and albumin [13]. None of the subjects had undergone dialysis or renal transplantation. ADPKD patients produced 1000–2000 ml of urine per day and proteinuria was negative or only trace. Each patient gave informed consent before the collection of body fluid samples.

Cyst fluid samples were generally obtained through percutaneous drainage of multiple renal cysts under ultrasonic guidance, and some through laparoscopic renal cyst decortication. The day before blood and urine samples were taken, the subjects ceased eating at 8 p.m. and drinking at 10 p.m. Peripheral blood and mid-stream urine samples were collected in the morning. Blood samples were treated with the anticoagulant agent sodium citrate and female urine samples were not harvested during menstruation. Blood was centrifuged at 3000 g for 10 min. Plasma was isolated and treated with Protease Inhibitor Cocktail Set (EMD Biosciences, Inc., San Diego, CA) and stored at –80°C. Fresh urine and cyst fluid were centrifuged at –80°C at 4000 g for 10 min, and the supernatant was treated with a protease inhibitor and stored at –80°C.

Human kidney specimens
Normal human renal tissue (n = 5) was obtained from kidneys surgically excised for a localized neoplasm. Tissue samples were obtained from macroscopically normal portions of kidney, located at some distance from the neoplastic process. ADPKD kidneys (n = 6) were excised for repeated haemorrhage. These patients were diagnosed and treated at Changzheng Hospital, Shanghai, China. The diagnosis of ADPKD was based on family medical history, ultrasound and computed tomography (CT) scan. Patients provided informed consent before tissue specimens were collected.

Tissues were fixed in 4% paraformaldehyde (for in situ hybridization) and 0.1% diethylpyrocarbonate (DEPC) or in 10% neutral buffered formalin (for immunohistochemistry) for ≥12 h, processed, paraffin embedded and sectioned at 3 µm. Additionally, some tissue samples from the nephrectomy specimens were snap frozen and utilized for RNA and protein preparation.

ELISA assays of SPARC levels in body fluids
The concentration of SPARC in body fluids was measured with an enzyme-linked immunosorbent assay (ELISA) kit (Hematologic Technologies, Essex Junction, VT). ELISAs were performed according to the manufacturer's instruction manual. The absorbance at 492 nm was determined using a microplate reader (Labsystems Dragon Wellscan MK3, Finland).

In situ hybridization
Oligonucleotide probes (5'-GCCTGGATCTTCTTTCTCCTTTGCCTGGCC-3', 5'-CTTCGACTCTTCCTGCCACTTCTTTGCCAC-3' and 5'-TGAAGAAGATCCATGAGAATGAGAAGCGCC-3') antisense to SPARC mRNA were synthesized and digoxigenein-UTP labelled at the 3'-terminus (Boehringer Mannheim, Germany). All the reagents and consumed materials used in RNA in situ hybridization processing were previously treated with 0.1% DEPC. The paraffin-embedded 3 µm thick sections were dewaxed in xylene and hydrated via a gradient of alcohol immersions. Endogenous peroxidase activity was blocked in 3% H2O2 at room temperature for 30 min and tissue was digested in proteinase K for 15 min to expose mRNA fragments. Sections were treated with pre-hybridization buffer [42% deionized formamide, 10% dextran sulfate, 0.1% DEPC, 0.5 mM phosphate-buffered saline (PBS)] at 38°C for 2 h. For subsequent hybridization, each section was then treated overnight with 20 µl of labelled probes (1.0 µg/ml) under a nuclease-free coverslip in a humid chamber at 38°C. Hybridized sections were washed and blocked and then incubated with biotinylated mouse anti-digoxigenin antibody, SABC-POD and biotinylated peroxidase. 3,3'-Diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich, MO) was used as the chromogen. The slides were counterstained with haematoxylin, dehydrated and coverslipped. Treatment with the same oligonucleotide probes without the digoxigenin label served as a negative control.

Immunohistochemistry
Tissue sections were dewaxed and endogenous peroxidase was inactivated. The sections were immersed in 0.01 M citrate buffer (pH 6.0), heated in a microwave oven to retrieve antigenicity and blocked for non-specific binding with 5% bovine serum albumin (BSA) in PBS for 20 min. Blocked sections were then incubated at 4°C overnight with mouse anti-SPARC monoclonal antibody (AON-5031, Hematologic Technologies). After washing in PBS, the sections were treated with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, CA) at 37°C for 20 min. Secondary antibody incubation was followed by the DAB colour development and counterstaining procedures described above. Incubation with an irrelevant non-immune mouse IgG primary antibody served as the negative control.

Western blot analysis
Western blot assays were performed with lysates from snap-frozen renal tissue samples. Human kidney tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% SDS, 5 µg/ml of aprotinin, 5 µg/ml of leupeptin). The protein concentration of each sample was determined by a Protein Assay Kit (Pierce Biotechnology, Inc., Rockford, IL). Lysates were fractionated on a 12% polyacrylamide gel that contained 0.1% SDS and then transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, USA) by electroblotting. The blots were blocked with TBST (10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween-20) containing 5% non-fat dry milk for 2 h at room temperature, and then incubated with 2 µg/ml monoclonal mouse anti-SPARC antibody (AON-5031) at 4°C overnight. The membrane was subjected to three 5 min TBST washes, and incubated at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG. After subsequent washing with TBST, signals were detected with enhanced chemiluminescence (Amersham, UK).

Cell culture
An ADPKD cyst-lining epithelial cell line was established in our laboratory [14]. A human kidney cortex (HKC) cell line was kindly provided by Dr Racusen from John Hopkins University. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (1 : 1) supplemented with 10% fetal bovine serum (Gibco) at 37°C with 5% CO2.

SPARC secretion assay
Secretion of SPARC into the culture medium was studied with cyst-lining epithelial cells and HKC cells. A total of 5 x 104 cells from each cell line were seeded into separate 25 cm2 cell culture flasks containing 5 ml of culture medium. Cell-free culture medium served as a control. After 3 days, culture medium (4 ml) was collected from each flask, and concentrated by superior ultrafiltration membranes (Millipore Corp., USA). The two kinds of cells were scraped and harvested. Total protein was extracted as described above for the western blot assay. Concentrated proteins from the media samples were used to detect SPARC levels, and the cellular protein extracts were used to detect GAPDH levels as an internal control for the relative amount of SPARC. The SPARC and GAPDH signal intensities were quantified by densitometric analysis.

Cell proliferation analysis
Cell proliferation was studied using bromodeoxyuridine (BrdU) incorporation ELISA (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. The exogenous human platelet SPARC was from Hematologic Technologies. ADPKD cyst-lining epithelial cells (5 x 103 cells/well) from the logarithmic growth phase were plated in three 96-well plates. When they had proliferated to 80% confluence, the cells synchronously entered a quiescent phase after the medium was changed to a serum-free medium for 24 h. After discarding the supernatant, the cells were treated with 10% fetal calf serum (FCS) medium containing SPARC at various concentrations (0, 1, 2.5, 5.0 and 10 µg/ml) for 48, 72 or 96 h. Each experimental group included six wells, and all experiments were repeated three times.

After incubation, the cells were labelled with BrdU (10 µmol/l) at 37°C for 6 h. DNA synthesis was assessed by measuring the amount of BrdU incorporation into the DNA, which was detected by immunoassay. Cellular proliferation was determined by measuring the magnitude of absorbance (A value) at 405 nm (reference wavelength 492 nm). The inhibition rate was expressed by the following equation:

Formula

Cell cycle analysis
Cell cycle phase was examined by fluorescence-activated cell sorting (FACS) analysis. Cyst-lining epithelial cells in a logarithmic growth phase were plated in 6-well plates and treated with SPARC at various concentrations (0, 1, 2.5, 5 and 10 µg/ml in media) for 72 h. The cells were trysinized, washed with PBS and fixed in 70% pre-cooled ethanol overnight at –20°C. After washing twice with PBS, the cells were stained with propidium iodide for FACS analysis in the dark. FACS analysis was performed under an excitation wavelength of 488 nm and a detection wavelength of 630 nm with Coulter Epics XL (Beckmen, USA). The FACS data were analysed by Multi-Cycle software (DNA Content and Cell Cycle Analysis Software, San Diego, CA). The proliferation index (PI%) was expressed by the following equation:

Formula

Analysis of apoptosis
Induction of apoptosis in cyst-lining epithelia after exogenous SPARC treatment was assessed by morphological observation under an inverted microscope (Olympus CK2) and use of an apoptosis index (AI). After 72 h of SPARC treatment, cells were harvested by trypsin digestion, fixed in 4% paraformaldehyde and 2% glutaraldehyde for ≥4 h and washed in PBS. Cell were clotted by addition of plasma, 1% osmium acid for 2–3 h, rinsing with PBS for 30 min and then embedded in Epon 812 at 37°C overnight. Ultrathin sections were stained with 4% uranyl acetate for 30 min, followed by washing, lead citrate staining for 5–10 min, washing again and drying. Ultrastructural changes in the epithelia were observed with a transmission electron microscope (H800, Hitachi, Japan). Cell preparation and analysis methods for FACS AI analysis were the same as that employed for cell cycle analysis, except that the samples included suspension cells in the media.

Real-time fluorescent quantitative RT–PCR
The mRNA level of SPARC in renal tissues was analysed by real-time fluorescent quantitative reverse transcription–polymerase chain reaction (RT–PCR). For further verification of SPARC effects on cell proliferation, cell cycle and apoptosis of cyst-lining epithelial cells, changes in mRNA levels of the cell proliferation-related genes (PCNA and MCM2), cell cycle-regulating genes (ClnD1 and p21Waf1) and apoptosis-related genes (Bax and Bcl-2) were analysed.

Total RNA was extracted from 50 mg of frozen renal tissue, and cells in a logarithmic growth phase were treated with 0 or 10 µg/ml SPARC for 72 h using Trizol according to the manufacturer's instructions (Life Technologies). Genomic DNA contaminants were removed with a DNase digest (TaKaRa Corp., Japan). Briefly, cDNA was synthesized from a total of 2 µg of RNA in a 20 µl reaction mixture that included 20 U of RNasin, 100 U of SS (Invitrogen), 4 µl of 5 x 1st Strand Buffer, 0.5 µM primers and 1 µl of 10 mM dNTP. The mixture was incubated at 25°C for 10 min, 42°C for 1 h and 52°C for 15 min. Reverse transcriptase was then inactivated by a 15 min incubation at 70°C. The primer sequences employed in RT–PCR are shown in Table 1. PCRs were carried out in 25 µl of reaction mixture [1.0 µl of cDNA, 2.5 µl of 10x buffer, 0.3 µl of 250 mM Mg2+, 0.3 µl of 25 mM dAGCU, 1.0 µl of 10 µM primers, 1.0 µl of 10–3x calibration buffer, 0.8 µl of 25x SYBR Green (BioRad Laboratories, Inc., Hercules, CA), 1.25 U of Taq polymerase and 0.4 U of UNG]. The following cycle conditions were used: pre-denaturing at 95°C for 90 s, five cycles of denaturation at 95°C for 5 s, annealing at 55°C for 15 s, extension at 72°C for 20 s; 35 additional denaturing cycles at 95°C for 5 s, annealing at 60°C for 30 s to reach the fluorescent signal detection point; another 40 cycles of denaturing at 95°C for 1 min, and annealing at 55°C for 1 min (increasing 0.5°C/cycle every 10 s). The amount of target gene expression was calculated from the respective standard curves, and quantitative expression was normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) with an iCycler Thermal Cycler (BioRad Laboratories, Inc., Hercules, CA).


View this table:
[in this window]
[in a new window]
  		Table 1. PCR primers

 
Statistical analysis
The data are presented as means±SD. Comparisons between results from different groups were performed using Student's t-test or one-way analysis of variance (ANOVA), as appropriate. Statistical significance was defined as P<0.05 in all cases. The software package was SAS 9.1.3.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The concentration of SPARC in plasma, urine and renal cyst fluid
The SPARC concentrations measured in the plasma, urine and renal cyst fluid samples of the ADPKD and SRC patients, and that in the plasma and urine samples of the normal subjects are summarized in Table 2. The influence of demographics such as age and sex was excluded. SPARC levels in renal cyst fluid of ADPKD patients (3628.75±1445.90 ng/ml) were greater than that in renal cyst fluid of SRC patients (154.14±41.28 ng/ml) (P<0.01). SPARC levels in urine of ADPKD patients (1253.16±544.81 ng/ml) were greater than that in urine of both SRC patients (134.08±30.00 ng/ml) and normal control subjects (123.91±28.37 ng/ml) (P<0.01); however, the mean SPARC level in urine of the SRC group did not differ from that of the normal control group (P>0.05). Plasma SPARC levels did not differ among the groups (ADPKD, 571.46±250.53; SRC, 494.86±173.30; and control, 496.71±169.98 ng/ml, P>0.05). SPARC levels in cyst fluid were greater than that in urine (P<0.01) for the ADPKD group, but not for the SRC group (P>0.05).


View this table:
[in this window]
[in a new window]
  		Table 2. The concentrations of SPARC in normal, SRC and ADPKD body fluids

 
According to the 2002 updates for the K/DOQI clinical practice guidelines for chronic kidney disease (Table 3), 14 of the ADPKD cases were classified as stage CKD1, nine as stage CKD2–3 and 14 as stage CKD4–5. With ADPKD disease stage progression, the patients’ renal function was aggravated gradually and the SPARC concentration in urine and cyst fluid tended to increase. However, SPARC concentrations in plasma, urine and cyst fluid did not differ among the patients of the different stages (P>0.05).


View this table:
[in this window]
[in a new window]
  		Table 3. The concentration of SPARC in plasma, urine and cyst fluid of the ADPKD group

 
The distribution of SPARC and its expression levels in renal tissues
In situ hybridization and immunohistochemistry were employed to detect SPARC distribution in normal and polycystic renal tissues. As shown in Figure 1, positive staining appeared as brown granules throughout the cytoplasm. Very weak expression of SPARC was observed in normal renal tubules. In renal tissues of adult patients with ADPKD, SPARC was localized mainly in cyst-lining epithelial cells, dilated tubules and collecting ducts, and presented strongly positive staining in cytoplasm.


Figure 1
View larger version (117K):
[in this window]
[in a new window]
  		Fig. 1. Localization of SPARC in normal adult kidneys and ADPKD kidneys by in situ hybridization (A and B) and immunohistochemistry (C and D). Only very weak SPARC staining appeared in renal tubules of normal adult renal tissues (A and C), but in ADPKD renal tissues (B and D) it was strongly positive in the cytoplasm of cyst-lining epithelial cells and dilated tubular cells.

 
SPARC mRNA and protein levels in normal and polycystic renal tissues were further compared with real-time fluorescent quantitative RT–PCR and western blot assays. The real-time fluorescent quantitative RT–PCR and western blot (Figures 2 and 3) experiments indicated that the SPARC mRNA level in ADPKD kidney tissue was (3.61±0.24) x 102 copies per million GAPDH, significantly higher than that in normal tissue (1.72±0.09)±102, and the SPARC protein level in ADPKD kidney tissue was also elevated relative to normal. The optical density ratio of SPARC protein and the corresponding GAPDH band in normal renal tissue and polycystic renal tissue were 44.68 and 81.25%, respectively. These results are consistent with our previous observation that SPARC is upregulated in ADPKD kidney tissue detected by cDNA expression microarray (unpublished findings).


Figure 2
View larger version (11K):
[in this window]
[in a new window]
  		Fig. 2. SPARC mRNA levels in kidney tissues. The SPARC mRNA level in normal adult kidneys was (1.72±0.09) x 102 copies per million GAPDH (internal control), whereas that in ADPKD kidneys was (3.61±0.24) x 102.

 

Figure 3
View larger version (67K):
[in this window]
[in a new window]
  		Fig. 3. Western blot analysis of SPARC in kidneys. The optical density values are denoted below each band.

 
Increased SPARC secretion in cyst-lining epithelial cells
Because SPARC is a secreted protein, it may affect growth of cyst-lining epithelia in an autocrine and/or paracrine manner. Therefore, SPARC secretion levels from HKC and cyst-lining epithelial cells were analysed. While western blot assay revealed no detectable SPARC from the concentrated protein in culture media of the control group, culture media from separate cultures of HKC cells or cyst-lining epithelial cells were found to have SPARC protein, and the level of the latter exceeded that of the former. The ratios of optical density values of SPARC protein secreted by HKC cells and cyst-lining epithelial cells to that of their corresponding GAPDH internal controls were 35.56 and 71.15%, respectively (Figure 4).


Figure 4
View larger version (57K):
[in this window]
[in a new window]
  		Fig. 4. Western blot analysis of SPARC in the culture media of HKC and cyst-lining epithelial (PKD) cells. Culture medium alone in a flask served as the control. GAPDH was detected to analyse the relative level of SPARC as an internal control. The optical density values are denoted below each band.

 
Exogenous SPARC inhibits the proliferation of cyst-lining epithelia and affects the mRNA levels of PCNA and MCM2
To investigate the autocrine/paracrine effects of SPARC on cyst-lining epithelial cell growth, epithelial cells were cultured for 48–96 h in medium containing different concentrations of exogenous human platelet SPARC. This result is important as it indicates whether platelet SPARC produces the same growth inhibitory effect on the cells used in this study as it does on other cells, e.g. endothelial, retinal pigment epithelial cells and ovarian cancer cells [15–17]. BrdU incorporation ELISA (described in Materials and methods) indicated that proliferation was progressively inhibited by treatment with increasing concentrations of SPARC. At 10 µg/ml, 48 h treatment with SPARC effectively inhibited proliferation of cyst-lining epithelial cells relative to controls (P<0.05). This inhibitory effect was time dependent and reached its peak at 96 h of treatment. After 96 h of SPARC treatment, all tested concentrations of SPARC were sufficient to inhibit cyst-lining epithelial cell proliferation relative to the control group (P<0.01) and relative to the same concentration after 48 and 72 h of treatment (P<0.05). The inhibition rate of SPARC at the maximum tested concentration of 10 µg/ml reached 49.19%. The above proliferation inhibition data are summarized in Table 4, and Figures 5 and 6.


View this table:
[in this window]
[in a new window]
  		Table 4. Concentration- and time-dependent manner of SPARC inhibition of cyst-lining epithelial cell proliferation

 

Figure 5
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 5. Inhibition rate of 10 µg/ml SPARC on cyst-lining epithelial cell proliferation at each time point. The inhibitory effect on epithelial cell proliferation increased in a time-dependent manner. The inhibition rates at 48, 72 and 96 h of SPARC treatment at 10 µg/ml were 28.64, 37.83 and 49.19%, respectively.

 

Figure 6
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 6. The inhibition rate of SPARC on cyst-lining epithelial cells at various concentrations. SPARC inhibition of cell proliferation increased gradually with increasing concentrations. After 96 h of SPARC treatment, the inhibition rates at 1, 2.5, 5 and 10 µg/ml were 31.23, 45.52, 48.55 and 49.19%, respectively.

 
The real-time fluorescent quantitative RT–PCR data describing SPARC treatment-induced changes in PCNA and MCM2 mRNA expression were consistent with those obtained from the BrdU incorporation ELISA. Treatment with 10 µg/ml SPARC for 72 h decreased the epithelial mRNA levels of both PCNA and MCM2. The mRNA level of PCNA decreased from (8.26±1.01) x 103 copies per million GAPDH to (3.58±1.16) x 103 (P<0.01), and MCM2 decreased from (5.23±1.01) x 103 to (1.39±0.54) x 103 (P<0.01) (Table 5).


View this table:
[in this window]
[in a new window]
  		Table 5. The effects of 10 µg/ml SPARC after 72 h of treatment on the expression of cell proliferation-related genes of cyst-lining epithelial cells

 
Exogenous SPARC inhibits cell cycle progression of cyst-lining epithelia and affects the mRNA levels of cell cycle regulatory genes
FACS was applied to evaluate the effects of SPARC on the cell cycle and PI% of cyst-lining epithelial cells. SPARC treatment for 72 h reduced the amount of cyst-lining epithelial cells in S + G2/M phases so that cells remained at G0/G1 longer, in a concentration-dependent manner. The PI% similarly gradually decreased with increasing SPARC treatment concentration (Table 6). Moreover, real-time fluorescent quantitative RT–PCR analysis revealed that 10 µg/ml SPARC treatment for 72 h decreased the mRNA level of ClnD1 from (7.50±0.99) x 104 copies per million GAPDH to (3.56±0.54) x 104 (P<0.01), but increased that of p21Waf1 from (4.25±1.38) x 103 to (7.72±0.85) x 103 (P<0.05) (Table 7).


View this table:
[in this window]
[in a new window]
  		Table 6. The effects of SPARC treatment (72 h) on cell cycle rate and proliferation indices (PIs) of cyst-lining epithelial cells

 

View this table:
[in this window]
[in a new window]
  		Table 7. The effects of 72 h SPARC treatment at 10 µg/ml on the expression of cell cycle regulation genes in cyst-lining epithelial cells

 
SPARC induces apoptosis in cyst-lining epithelia and affects the mRNA levels of cell apoptosis-related genes
Recent studies have confirmed that there are imbalances of proliferation and apoptosis in ADPKD cystic cells [18]. Thus, to determine whether exogenous SPARC can induce cyst-lining epithelial cell apoptosis, SPARC treatment effects on cyst-lining epithelial cell morphology were assessed and their AIs were measured. After 48 h treatment with SPARC at 2.5, 5 and especially 10 µg/ml, some previously irregularly shaped cells became elliptical or round and some became suspended with intact cellular membranes from adherent cells. Examination by electron microscopy revealed typical apoptotic changes with SPARC treatment, i.e. numerous cytoplasmic vacuoles and fissures emerged, nuclei became irregular with concentrated skirted chromatin and typical apoptotic bodies were observed in some cells (Figure 7).


Figure 7
View larger version (128K):
[in this window]
[in a new window]
  		Fig. 7. Ultrastructural changes of SPARC-treated cyst-lining epithelial cells. (A) Normal structure of control cells. (B) SPARC at 2.5 µg/ml. Note the numerous cytoplasmic vacuoles (arrow e). (C) SPARC at 5 µg/ml. Many cytoplasmic fissures (arrow f) are apparent. (D) SPARC at 10 µg/ml. Concentrated and skirted chromatin (arrow g) and typical apoptotic bodies (arrow h) in cytoplasm are visible.

 
Apoptotic peaks and AIs were detected by FACS after 48 h of 2.5, 5 and 10 µg/ml SPARC treatment. As shown in Figure 8, the typical hypodiploid peak (blue) was observed in the DNA histograms of all groups. The area of the apoptotic peak increased with increased SPARC treatment concentration. The corresponding AIs of all groups were 8.2, 12.7, 13.3 and 29.5%, respectively.


Figure 8
View larger version (87K):
[in this window]
[in a new window]
  		Fig. 8. Apoptosis index of cyst-lining epithelial cells after SPARC treatment with 0 (A, control), 2.5 (B), 5 (C) and 10 (D) µg/ml.

 
Real-time fluorescent quantitative RT–PCR measurements of changes in the apoptosis-related genes Bax and Bcl-2 after SPARC treatment at 10 µg/ml for 72 h revealed a selective effect of the treatment on Bcl-2. While the mRNA level of Bax was not affected, Bcl-2 was decreased from (8.05±0.72) x 103 to (6.50±0.80) x 103 copies per million GAPDH (P<0.05). This selective decrease in Bcl-2 resulted in a decreased Bcl-2 to Bax ratio (1.67±0.09 vs 2.06±0.09, P<0.05) (Table 8).


View this table:
[in this window]
[in a new window]
  		Table 8. The effects of 72 h SPARC treatment at 10 µg/ml on the expression of apoptosis-related genes in cyst-lining epithelial cells

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Elevated SPARC in ADPKD
Recent studies have indicated that SPARC has antitumour activity. Notably, tumour growth and metastasis in several tumour models were enhanced in SPARC gene knockout mice [19], and tumour-suppressing properties of SPARC have been reported in in vitro studies with ovarian cancer cells and neuroglioma cells [17,20]. The present study of the distribution and expression of SPARC in body fluids and renal tissues of ADPKD patients complements previous research on SPARC's antitumour activity and is consistent with our previous observation (unpublished findings) that SPARC is upregulated in ADPKD kidney tissues. The present findings indicate that SPARC is expressed strongly and mainly in cyst-lining epithelial cells and dilated renal tubules of ADPKD patients. In addition, we found that SPARC levels were elevated in urine of ADPKD patients relative to SRC patients and normal subjects. SPARC levels in cyst fluid for ADPKD patients was markedly higher than that in urine.

SPARC is a secreted glycoprotein with a molecular weight of 32 kDa after cleavage of the signal sequence. Micromolecular proteins with a molecular weight of 32 kDa in urine would be expected to be derived from glomerular filtration. However, in this study, the ADPKD patients were negative or only mildly positive on urine protein tests. Moreover, SPARC mRNA and protein were found to be located in cyst-lining epithelial cells, dilated renal tubules and renal collecting ducts. Previous studies have shown that SPARC is expressed in the renal tubular epithelium, collecting duct, urethra and bladder epithelium [21]. Therefore, SPARC in ADPKD patients’ urine is probably from the urine outflow duct including the renal tubule, collecting duct, bladder and urethra. SPARC originating from the kidney is probably derived mainly from the renal tubule and collecting system, as well as the epithelial cells of little cysts along the renal tubule and collecting duct. Because the kidney is the main organ affected in ADPKD and neither the bladder nor the urethra were found to have pathological changes in our ADPKD patients, we did not include an analysis of the urethra and bladder in this study. Our findings suggest that the markedly elevated SPARC concentration in the urine of patients with ADPKD relative to that in normal subjects and patients with SRC is due to increased SPARC secretion from dilated renal tubules and epithelial cells of renal tubule cysts.

Our findings with cultured cyst-lining epithelial cells and normal renal tubular cell supernatant are consistent with this view. As ADPKD develops, little cysts associated with the collecting ducts gradually lose contact as the ducts dilate. Therefore, SPARC in renal cyst fluid may be derived mainly from cyst-lining epithelial cells. Moreover, the finding that cyst-lining epithelial cells secreted more SPARC than tubular cells in vitro may explain, at least in part, the higher SPARC levels found in the cyst fluid of ADPKD patients compared with that in urine and the obvious difference in cyst fluid SPARC levels between the ADPKD group and SRC group. However, as the present study included relatively few SRC patients and histological data about SPARC in the renal tissue of SRC patients were not obtained, it is not known whether cyst fluid SPARC levels would differ between large samples of SRC and ADPKD patients.

Besides, we did find that urine and cyst fluid SPARC levels had a trend to increase along with the progression of renal function aggravation. However, this effect was not statistically significant in the present study. Therefore, further research focused on this question with larger group sizes will be required to determine whether there is a real relationship between SPARC levels in urine and cyst fluid and deterioration of renal function.

SPARC inhibition of cyst-lining epithelial cell proliferation and the expression of PCNA and MCM2
Recent studies have demonstrated that the cyst-lining epithelial cells of a polycystic kidney have the characteristic of high multiplication similar to that seen in benign tumours, which can promote the emergence and dilation of more cysts [22]. The antiproliferative activity of SPARC has been demonstrated in endothelial cells [23]. Domain IV of SPARC, which contains an EF-hand-like loop and has a high-affinity Ca2+-binding site, is sufficient to induce SPARC-mediated growth inhibitory functions [24]. Furthermore, mesangial cells, fibroblasts and smooth muscle cells isolated from SPARC-null mice were found to grow faster than their respective wild-type counterparts [25].

The present study provides an in vitro demonstration that exogenous SPARC can reduce epithelial cell proliferation in a concentration- and time-dependent manner. Microgram concentrations of SPARC inhibited cyst-lining epithelial cell proliferation and markedly reduced PCNA and MCM2 mRNA expression. PCNA is a nuclear protein that is expressed specifically during cell proliferation. Its expression starts at the end of G1 phase, reaches its peak during S phase and early G2 phase, and ceases during M and G0 phases [26]. MCM2 has a key role in DNA replication. The expression of MCM mRNAs and proteins selectively increases during proliferation and this increase is proportional to the degree of cell proliferation [27]. These findings suggest that SPARC may also inhibit cyst-lining epithelial cell proliferation in vivo.

SPARC regulation of the cell cycle via influences on ClnD1 and p21Waf1 expression
SPARC treatment increased the number of cells at G0/G1 phase and decreased the number of cells at S phase to a similar degree, indicating that SPARC inhibition of cell proliferation may be mediated through a regulatory effect on the G1 phase. The cell cycle is the final pathway for cell proliferation and has several checkpoints. The G1–S checkpoint is key because this is when cells integrate a convergence of signals to determine whether to initiate cell division, to enter into a resting state (G0 phase) or to undergo apoptosis.

Cyclin D1 encoded by ClnD1 is an important cyclin associated with G1 phase regulation that can control cyclin-dependent kinase (CDK) activity by phosphorylation and enhance the expression of some genes whose products promote the passage of cells through the G1–S checkpoint and hence the commencement of cell division [28]. The first CDK inhibitor identified in mammalian cells, p21Waf1, suppresses the activity of CDK or the cyclin D1–CDK complex through dephosphorylation and inhibits cell proliferation [29]. PKD1 can activate the JAK–STAT pathway, thereby upregulating p21Waf1 and inducing cell cycle arrest in G0/G1. Increased expression of p21Waf1 is primarily responsible for mediating the growth-suppressing effects of PKD1 in an experimental system [30]. In this study, we found that SPARC treatment decreased ClnD1 mRNA levels but increased p21Waf1 mRNA levels in cyst-lining epithelial cells. These data suggest that SPARC may regulate the cell cycle in cyst-lining epithelial cells by concurrently inhibiting ClnD1 expression and augmenting p21Waf1 expression. By this mechanism, SPARC may prevent cyst-lining epithelial cells from entering into S phase and keep them in a resting state.

SPARC regulation of the apoptotic regulatory factors Bcl-2 and Bax
Both epithelial cell apoptosis and proliferation are dysregulated in ADPKD [31,32]. Dysregulation of the balance between pro- and anti-apoptotic Bcl-2 family members correlates with increased apoptosis in ADPKD [33]. The ratio of Bcl-2–Bax heterodimers to Bax–Bax homodimers is a critical factor in determining apoptosis susceptibility [34]. We found that SPARC treatment could induce cyst-lining epithelial cell apoptosis and simultaneously reduce the ratio of Bcl-2 to Bax. Therefore, the regulation of Bcl-2 and Bax expression may be a key mechanism underlying SPARC induction of apoptosis in cyst-lining epithelial cells.

Putative SPARC receptors
The antiproliferative and apoptotic effects of exogenous SPARC on cyst-lining epithelial cells are consistent with the presence of cell surface receptors for SPARC. This hypothesis is supported by the results obtained from studying the mediators through which exogenous SPARC exerts its counteradhesive and antiproliferative effects on endothelial cells. Pre-treating endothelial cells with protein tyrosine kinase inhibitors protected them against the inhibitory effect of SPARC on cell spreading. Moreover, inhibition of cell cycle progression by SPARC was blocked by treatment with heterotrimeric G protein inhibitors such as pertussis toxin and cholera toxin [35]. A recent study showed the first direct evidence that SPARC binds to putative SPARC receptors on the cell surfaces of HOSE and ovarian cancer cells using a fusion protein containing SPARC and human placental alkaline phosphatase [19]. To date, however, neither a putative SPARC receptor nor an intracellular signalling pathway triggered by SPARC has been identified.

Summary
In this study, we showed that exogenous SPARC could reduce proliferation and induce apoptosis in cyst-lining epithelial cells. The antiproliferative effect may be mediated by an inhibition of ClnD1 and stimulation of p21Waf1 expression, thereby preventing the passage of cells through the G1–S checkpoint. SPARC may also induce apoptosis of epithelial cells by decreasing the ratio of Bcl-2 to Bax gene expression. The findings of the SPARC distribution in kidney tissues and in ADPKD patient body fluids indicate that SPARC may play a role in negative feedback. All these suggest that SPARC may have a protective effect in ADPKD.



   Acknowledgments
 
This work was supported by grants from the National 863 Plan in High Technology Progress (2002AA2Z3130) and the National Nature and Science Foundation (30330640).

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Wilson PD. Polycystic kidney disease. N Engl J Med 2004; 350: 151–164[Free Full Text]
  2. Somlo S, Markowitz GS. The pathogenesis of autosomal dominant polycystic kidney disease: an update. Curr Opin Nephrol Hypertens 2000; 9: 385–394[CrossRef][ISI][Medline]
  3. Wu G. Current advances in molecular genetic of autosomal-dominant polycystic kidney disease. Curr Opin Nephrol Hypertens 2001; 10: 23–31[CrossRef][ISI][Medline]
  4. Gabow PA, Johnson AM, Kaehny WD et al. Factors affecting the progression of renal disease in autosomal-dominant polycystic kidney disease. Kidney Int 1992; 41: 1311–1319[ISI][Medline]
  5. Torres VE. Extrarenal manifestations of autosomal dominant polycystic kidney disease. Am J Kidney Dis 1999; 34: xlv–xlviii[Medline]
  6. Trudel M, Guillaume R. Progress in molecular genetics of autosomal dominant polycystic kidney disease. Front Biosci 2000; 5: D312–D320[Medline]
  7. Yan Q, Sage EH. SPARC, a matricellular glycoprotein with important biological functions. J Histochem Cytochem 1999; 47: 1495–1505[Free Full Text]
  8. Bornstein P. Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1. J Cell Biol 1995; 130: 503–506[Free Full Text]
  9. Sage EH. Regulation of interactions between cells and extracellular matrix: a command performance on several stages. J Clin Invest 2001; 107: 781–783[ISI][Medline]
  10. Francki A, Sage EH. SPARC and the kidney glomerulus: matricellular proteins exhibit diverse functions under normal and pathological conditions. Trends Cardiovasc Med 2001; 11: 32–37[CrossRef][ISI][Medline]
  11. Francki A, Motamed K, McClure TD et al. SPARC regulates cell cycle progression in mesangial cells via its inhibition of IGF-dependent signaling. J Cell Biochem 2003; 88: 802–811[CrossRef][Medline]
  12. National Kidney Foundation K/DOQI Clinical Practice Guidelines for Chronic Kidney Disease: Evaluation, Classification and Stratification. Am J Kidney Dis [Suppl 1] 2002; 37: S182–S238
  13. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Ann Intern Med 1999; 130: 461–470[Abstract/Free Full Text]
  14. Xu CG, Mei CL, Zhang LM. Establishment and identification of ADPKD cyst-lining epithelia cell lines. J Nephrol Dial Transplant 2000; 9: 142–145
  15. Motamed K, Blake DJ, Angello JC et al. Fibroblast growth factor receptor-1 mediates the inhibition of endothelial cell proliferation and the promotion of skeletal myoblast differentiation by SPARC: a role for protein kinase A. J Cell Biochem 2003; 90: 408–423[CrossRef][Medline]
  16. Hagan S, Hiscott P, Sheridan CM, Wong D, Grierson I, McGalliard J. Effects of the matricellular protein SPARC on human retinal pigment epithelial cell behavior. Mol Vis 2003; 9: 87–92[Medline]
  17. Yiu GK, Chan WY, Ng SW et al. SPARC (secreted protein acidic and rich in cysteine) induces apoptosis in ovarian cancer cells. Am J Pathol 2001; 159: 609–622[Abstract/Free Full Text]
  18. Gogusev J, Murakami I, Doussau M et al. Molecular cytogenetic aberrations in autosomal dominant polycystic kidney disease tissue. J Am Soc Nephrol 2003; 14: 359–366[Abstract/Free Full Text]
  19. Brekken RA, Puolakkainen P, Graves DC, Workman G, Lubkin SR, Sage EH. Enhanced growth of tumors in SPARC null mice is associated with changes in the ECM. J Clin Invest 2003; 111: 487–495[CrossRef][ISI][Medline]
  20. Vadlamuri SV, Media J, Sankey SS, Nakeff A, Divine G, Rempel SA. SPARC affects glioma cell growth differently when grown on brain ECM proteins in vitro under standard versus reduced-serum stress conditions. Neuro Oncol 2003; 5: 244–254[Abstract]
  21. Alpers CE, Hudkins KL, Segerer S et al. Localization of SPARC in developing, mature, and chronically injured human allograft kidneys. Kidney Int 2002; 62: 2073–2086[CrossRef][ISI][Medline]
  22. Peter I, Stefan S. Genetics and pathogenesis of polycystic kidney disease. J Am Soc Nephrol 2002; 13: 2384–2398[Free Full Text]
  23. Sage EH. Secretion of SPARC by endothelial cells transformed by polyoma middle T oncogene inhibits the growth of normal endothelial cells in vitro. Biochem Cell Biol 1992; 70: 579–592[ISI][Medline]
  24. Kupprion C, Motamed K, Sage EH. SPARC (BM-40, osteonectin) inhibits the mitogenic effect of vascular endothelial growth factor on microvascular endothelial cells. J Biol Chem 1998; 273: 29635–29640[Abstract/Free Full Text]
  25. Bradshaw AD, Francki A, Motamed K, Howe C, Sage EH. Primary mesenchymal cells isolated from SPARC-null mice exhibit altered morphology and rates of proliferation. Mol Biol Cell 1999; 10: 1569–1579[Abstract/Free Full Text]
  26. Takahashi H, Oishi Y, Chuang SS, Morii S. Effects of tissue fixation and processing on proliferating cell nuclear antigen (PCNA) immunohistochemistry. Acta Pathol Jpn 1992; 42: 621–623[Medline]
  27. Lindner K, Gregan J, Montgomery S, Kearsey SE. Essential role of MCM proteins in premeiotic DNA replication. Mol Biol Cell 2002; 13: 435–444[Abstract/Free Full Text]
  28. Wang C, Li Z, Fu M, Bouras T, Pestell RG. Signal transduction mediated by cyclin D1: from mitogens to cell proliferation: a molecular target with therapeutic potential. Cancer Treat Res 2004; 119: 217–237[Medline]
  29. Fotedar R, Bendjennat M, Fotedar A. Functional analysis of CDK inhibitor p21WAF1. Methods Mol Biol 2004; 281: 55–72[Medline]
  30. Bhunia AK, Piontek K, Boletta A et al. PKD1 induces p21(waf1) and regulation of the cell cycle via direct activation of the JAK–STAT signaling pathway in a process requiring PKD2. Cell 2002; 109: 157–168[CrossRef][ISI][Medline]
  31. Lanoix J, D’Agati V, Szabolcs M, Trudel M. Dysregulation of cellular proliferation and apoptosis mediates human autosomal dominant polycystic kidney disease (ADPKD). Oncogene 1996; 13: 1153–1160[ISI][Medline]
  32. Ecder T, Melnikov VY, Stanley M et al. Caspases, Bcl-2 proteins and apoptosis in autosomal-dominant polycystic kidney disease. Kidney Int 2002; 61: 1220–1230[CrossRef][ISI][Medline]
  33. Ecder T, Melnikov VY, Stanley M et al. Caspases, Bcl-2 proteins and apoptosis in autosomal-dominant polycystic kidney disease. Kidney Int 2002; 61: 1220–1230[CrossRef][ISI][Medline]
  34. Poeta GD, Venditti A, Principe MI et al. Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML). Blood 2003; 101: 2125–2131[Abstract/Free Full Text]
  35. Motamed K, Sage EH. SPARC inhibits endothelial cell adhesion but not proliferation through a tyrosine phosphorylation-dependent pathway. J Cell Biochem 1998; 70: 543–552[CrossRef][ISI][Medline]
Received for publication: 19. 6.05
Accepted in revised form: 8.12.05


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
M. J. Tang and I. T. Tai
A Novel Interaction between Procaspase 8 and SPARC Enhances Apoptosis and Potentiates Chemotherapy Sensitivity in Colorectal Cancers
J. Biol. Chem., November 23, 2007; 282(47): 34457 - 34467.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
F. Taghizadeh, M. J. Tang, and I. T. Tai
Synergism between vitamin D and secreted protein acidic and rich in cysteine-induced apoptosis and growth inhibition results in increased susceptibility of therapy-resistant colorectal cancer cells to chemotherapy
Mol. Cancer Ther., January 1, 2007; 6(1): 309 - 317.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
21/5/1278    most recent
gfk036v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Wang, W.
Right arrow Articles by Dai, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, W.
Right arrow Articles by Dai, B.
Social Bookmarking