Medulloblastomas overexpress the p53-inactivating oncogene WIP1/PPM1D

Medulloblastoma is the most common malignant brain tumor of childhood. Despite numerous advances, clinical challenges range from recurrent and progressive disease to long-term toxicities in survivors. The lack of more effective, less toxic therapies results from our limited understanding of medulloblastoma growth. Although TP53 is the most commonly altered gene in cancers, it is rarely mutated in medulloblastoma. Accumulating evidence, however, indicates that TP53 pathways are disrupted in medulloblastoma. Wild-typep53-induced phosphatase 1 (WIP1 or PPM1D) encodes a negative regulator of p53. WIP1 amplification (17q22-q23) and its overexpression have been reported in diverse cancer types. We examined primary medulloblastoma specimens and cell lines, and detected WIP1 copy gain and amplification prevalent among but not exclusively in the tumors with 17q gain and isochromosome 17q (i17q), which are among the most common cytogenetic lesions in medulloblastoma. WIP1 RNA levels were significantly higher in the tumors with 17q gain or i17q. Immunoblots confirmed significant WIP1 protein in primary tumors, generally higher in those with 17q gain or i17q. Under basal growth conditions and in response to the chemotherapeutic agent, etoposide, WIP1 antagonized p53-mediated apoptosis in medulloblastoma cell lines. These results indicate that medulloblastoma express significant levels of WIP1 that modulate genotoxic responsiveness by negatively regulating p53.
Introduction
Medulloblastoma is the most common malignant brain tumor of childhood [1, 2]. Treatment with surgery, radiation, and chemotherapy successfully cures many patients, but survivors can suffer significant long-term toxicities affecting their neurocognitive and growth potential [3]. Despite clinical advances, up to 30% of children with medulloblastoma experience tumor progression or recurrence, for which no curative therapy exists. The lack of more effective, less toxic therapies stems from our imperfect understanding of the molecular processes that underlie medulloblastoma growth.
Among the most common cytogenetic lesions affecting medulloblastoma are the gain of the long arm of chromosome 17 (17q) and isochromosome 17q (i17q), consisting of 17p deletion with duplication of 17q, in approximately one-third of cases [4, 5]. Because i17q has been described as the sole cytogenetic lesion in certain medulloblastomas, it may represent a primary event rather than an alteration associated with clonal evolution [4]. Investigators have long sought oncogenes on 17q and tumor suppressor genes on 17p, but with limited success [6].
Although the TP53 tumor suppressor gene (17p13.1) is mutated in approximately half of human malignancies, it is rarely mutated in medulloblastoma [7–9]. However, several lines of evidence suggest that the p53 pathway is perturbed in medulloblastoma. Frank et al. have described activation of the p53-p14ARF pathway in the large cell/anaplastic variant of medulloblastoma [10, 11]. Our collaborators have noted significant nuclear p53 immunopositivity consistent with its activation in approximately 50% of primary human medulloblastoma examined (Adekunle Adesina, personal communication). Deletion of the murine homolog, Trp53, in the Patched haploinsufficient (Ptch+/−) mouse model increases the incidence of spontaneous medulloblastoma from approximately 15 to 100% [12]. Individuals with Li–Fraumeni syndrome who carry germline TP53 mutations are at increased risk for developing medulloblastoma, but fewer than 10% of sporadic medulloblastoma display TP53 mutations [4, 5].
The activity of p53 may be abrogated by alternate mechanisms as observed in other cancers with wild-typeTP53 [7]. One mechanism by which p53 function is limited is through amplification or overexpression of MDM2. MDM2 encodes an E3 ubiquitin ligase that normally functions in a negative feedback loop to down-regulate p53 expression, and is amplified in approximately 7% of human malignancies that lack mutation of TP53 [13]. However, Adesina et al. and Batra et al. have documented the absence of MDM2 amplification in medullobastoma [14, 15]. Although MDM2 overexpression has been detected in adult patients [16], there is no functional data to implicate MDM2 in p53 inactivation in medulloblastoma, suggesting alternative mechanisms.
The p53-induced proto-oncogene, WIP1 (wild-typep53-induced phosphatase 1 or protein phosphatase, magnesium-dependent 1, delta, PPM1D) maps to 17q22-q23. Amplification of 17q22-q23 has been described in several cell lines and malignancies including neuroblastoma, breast and ovarian carcinomas, most of which are wild-type for TP53 [17–19]. WIP1 displays p53-dependent oncogenic properties by inactivating p53, p38MAPK, and ATM pathways [17–21]. Previous reports of genomic analysis of medulloblastoma have implicated the gain of the WIP1 locus in its overexpression in a subset of tumors [22, 23].
We present data that confirm prior observations and also define the effects of WIP1 on medulloblastoma cell growth. We have quantitated WIP1 overexpression in primary medulloblastoma specimens and cell lines previously analyzed by comparative genomic hybridization (CGH) [24]. Using fluorescence in situ hybridization (FISH), we determined that WIP1 copy gain and amplification is prevalent among but not exclusively found in the tumors with 17q gain and i17q. We surveyed 33 primary medulloblastoma specimens for WIP1 expression and determined that RNA levels were significantly higher in 16 tumors with 17q gain or i17q. Similarly, our Western immunoblot analysis confirmed significant levels of WIP1 protein expression in primary tumors, generally higher in those with 17q gain or i17q. Genotoxic stress induces p53 phosphorylation and WIP1 expression in D283Med and Daoy medulloblastoma cell lines. Transient transfections indicate that WIP1 antagonizes p53-mediated cell cycle arrest and apoptosis in response to the chemotherapeutic agent, etoposide. These results support the hypothesis that medulloblastoma express significant levels of WIP1 that modulate genotoxic responsiveness by negatively regulating p53.
Materials and methods
Medulloblastoma tissue and cell lines
Medulloblastoma samples (n = 33) were obtained from children diagnosed at Texas Children’s Hospital, Houston, TX, between 1996 and 2004, following informed consents. Tissues were snap-frozen and stored in liquid nitrogen until processed. All specimens were obtained at the time of diagnosis prior to radiation or chemotherapy and subjected to histopathologic review according to WHO criteria [1]. Seven tumors displayed classic histology, 11 were uniformly desmoplastic, nodular, or well-differentiated, seven were predominantly anaplastic or large cell, an additional seven displayed some degree of anaplasia or large cell histology, and one was regarded as none of the above.
Ten patients were female and 23 were male. The mean age at diagnosis was 82 months (median 79 months) with four patients less than 36 months old (range: 12–216 months). The median and mean time of follow-up was approximately 42 months from diagnosis. The majority of patients were M0 (non-metastatic), while one was M2 and eight were M3 stage at diagnosis. All studies were performed according to the institutional guidelines of Texas Children’s Hospital, and with the approval of the Institutional Review Board of Baylor College of Medicine.
The breast carcinoma cell line MCF-7 with known high-level WIP1 amplification, the un-amplified breast epithelial cell line MCF-10A, and the medulloblastoma cell lines, Daoy and D283Med (American Type Culture Collection, Manassas, VA) were maintained as previously described in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) with high glucose (6 g/l), 2 mM l-glutamine and 10% (vol/vol) heat-inactivated fetal calf serum (Invitrogen, Carlsbad, CA) at 37°C in 5% CO2. Etoposide (VP-16) and cisplatin (CDDP) were applied to cell cultures as media-diluted stock solutions in PBS and DMSO, respectively (Sigma, St. Louis, MO). Ultraviolet (UV) irradiation was performed by exposing cell cultures using a GS Gene Linker UV Chamber (Bio-Rad Laboratories, Hercules, CA).
Fluorescence in situ hybridization (FISH) analysis
Primary medulloblastoma cells were harvested from first passage cultures. Chromosomes were prepared according to standard methods, including confirmatory karyotyping with G-banding [25]. Eleven first or second passage cultures of primary tumors were harvested when cultures were still sub-confluent. The BAC containing the WIP1 locus at chromosome 17q23 (RP11-67D12) and a plasmid containing chromosome 17 centromeric sequences (pZ17-14, generously provided by Dr. Mariano Rocchi) were fluorescently labeled with Spectrum Green and Spectrum Orange, respectively (Vysis, Downers Grove, IL), by nick translation and hybridized to metaphase or interphase spreads of cell lines (MCF-7, Daoy, D283) and primary tumor specimens [26]. We confirmed the map position of WIP1 on normal human metaphase spreads by FISH, as described previously [27]. The slides were counterstained with DAPI, and the images were captured using the Quips Pathvysion System (Applied Imaging, Santa Clara, CA). To determine the WIP1 copy number status, we analyzed 100–200 individual interphase nuclei for each case. The presence of copy gain or amplification was indicated if more than 10% of tumor nuclei displayed an increased copy number relative to the chromosome 17 centromeric probe signals and/or tumor ploidy. In cases in which there was a discrepancy between the number of centromeric signals and ploidy, the ploidy number was used to estimate the relative copy number.
Quantitative real-time RT-PCR (qRT-RTPCR) analysis
Total cellular RNA was prepared from frozen tumor tissue with either Trizol (Invitrogen) or an RNeasy kit (Qiagen, Valencia, CA) depending upon relative abundance, according to the manufacturers’ recommendations. Integrity and concentration of RNA were verified on the Baylor College of Medicine, Houston, TX Microarray Core Facility Bioanalyzer (Agilent Technologies, Santa Clara, CA). Total cellular RNA was reverse transcribed with SuperScript First-Strand Synthesis (Invitrogen) primed with oligo-(dT)12. PCR reactions were performed in a Bio-Rad iCycler using iQ Syber Green Supermix (Bio-Rad Laboratories), and primers listed below. Each PCR reaction was performed for 40 cycles, and performed in triplicate. Primer sequences included: WIP1 sense, 5′-ATCCGCAAAGGCTTTCTCGCTT-3′ and antisense, 5′-TTGGCCATTCCGCCAGTTTCTT-3′; TP53 sense, 5′-CCATCTACAAGCAGTCACAGC-3′ and antisense, 5′-GAGTCTTCCAGTGTGAGATG-3′; GAPDH sense, 5′-AAGGTGAAGGTCGGAGTCAA-3′ and antisense, 5′-AATGAAGGGGTCATTGATGG-3′. Expression of target RNA was internally normalized to expression of GAPDH gene and expressed relative to target gene expression in a human fetal brain RNA pool (Stratagene, La Jolla, CA). Human adult cerebellar (Stratagene) and human fetal cerebellar RNA samples (BioChain, Hayward, CA) were also assayed to provide tissue controls. Amplification products were verified by analyzing melting curves, agarose gel electrophoresis, and with direct sequencing of PCR products.
Western immunoblot analysis
Protein was extracted from frozen tissue by solubilization in boiling lysis buffer (0.5% SDS, 50 mM Tris–Cl, pH 8, 5 mM Na2EDTA, 2% β-mercaptoethanol). Lysates were stored at −80°C and separated by SDS-PAGE, transferred to nitrocellulose membranes (Pall Life Sciences, Ann Arbor, MI), and immunoblotted using standard methods. Primary antibodies included: WIP1 monoclonal antibody (Trevigen, Gaithersburg, MD), Phospho-p53 serine-15 (Ser15)-specific monoclonal antibody (Cell Signaling Technology, Danvers, MA), p53 monoclonal antibody (DO-1, Santa Cruz Biotechnology, Santa Cruz, CA), and β-Actin monoclonal antibody (Sigma). Staining with secondary antibody (Alexa Fluor 680-nm-conjugated goat anti-mouse IgG, Invitrogen) was visualized using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, Nebraska). Intensity of immunostaining was quantitated using ImageQuant 5.2 (Molecular Dynamics, Piscataway, NJ). Expression of target proteins in each sample was internally normalized to β-actin (determined on re-probed blots), relative to expression in the MCF-10A cell line.
Transfections and luciferase reporter assays
Cell lines were transiently transfected with expression and reporter plasmids using Lipofectin according to manufacturer’s recommendations (Invitrogen). Expression plasmids included: the human cytomegalovirus (CMV) immediate-early (IE) promoter-driven pP53-EGFP plasmid, encoding a p53-enhanced green fluorescent protein (GFP) fusion protein (Clontech, Mountainview, CA). Reporter plasmids included the pP21-luciferase construct to detect p53-activated transcription [28] and the actin promoter-driven Renilla luciferase plasmid, pB-Actin-RL, to provide transfection controls [29]. In each transfection, the expression and reporter plasmids were supplemented by empty vector plasmid (pcDNA3.1, Invitrogen) to achieve an equivalent total DNA concentration.
For flow cytometry, the EGFP-expressing pMaxGFP plasmid (Amaxa, Gaithersburg, MD) provided transfection controls. For functional assessment of in vitro p53 activation, cells transfected with luciferase-encoding reporter plasmids were harvested and processed for dual color luciferase assay according to manufacturer’s recommendations (Promega, Madison, WI), and quantitated using a BenchMark Plus plate spectrophotometer (Bio-Rad).
Flow cytometric and terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick end-labeling (TUNEL) analysis
We monitored DNA-indices for cell cycle analysis by multi-parametric flow cytometry using standard methods. Analyses were performed using a Becton Dickinson FACScan flow cytometer (BD Biosciences, San Jose, CA) for the detection of cells stained with propidium iodide (PI) and a 488 nm laser with filter combination for fluorescein isothiocyanate (FITC) and GFP. Single cell suspensions were isolated from culture, fixed in methanol, and stained with PI (100 μg/ml in PBS). Each histogram represents 10,000–100,000 cells for measuring DNA-index and cell cycle. Histogram analysis was performed with the CellQuest program (BD Biosciences). We calculated the sub-G0/G1 peak at a position in the hypodiploid area below a DNA-index of one (2n). Because the nucleus becomes fragmented during apoptosis and numerous individual chromatin fragments may be present in a single cell, the percentage of objects with a fractional DNA-content is represented by the sub-G0/G1 peak. Apoptotic nuclei were identified as a sub-diploid DNA peak, and were distinguished from cell debris on the basis of forward light scatter and PI fluorescence. The significance of differences in apoptotic populations was determined using paired Student t-test.
To detect apoptotic nuclei in sub-G0/G1 peaks, a subset of unstained cells was analyzed for DNA-fragmentation using the Frag-EL DNA fragmentation detection kit, according to manufacturer’s recommendations (Oncogene Research Products, Cambridge, MA). Cells were also counter-stained with PI for DNA quantification. FITC signal was detected on one channel in the logarithmic mode, while UV fluorescence (PI) was recorded in the linear mode on a separate channel. For each measurement, at least 10,000 cells were analyzed. We used CellQuest software for multi-parametric calculations and analyses. Cutoff negative and positive cells resulted from FITC-fluorescence isotype control measurements for each sample.
Results
WIP1 DNA copy number is increased in medulloblastomas with 17q gain
We employed FISH to analyze copy numbers of the WIP1 gene in primary medulloblastoma specimens and cell lines previously analyzed by CGH (Fig. 1). Compared to the centromeric chromosome 17 probe, which consistently displayed 2–4 signals in each metaphase or interphase nucleus, the 17q23 BAC probe (RP11-67D12) containing the WIP1 locus revealed a wider range of copy numbers. By FISH, we identified high-level amplification of the WIP1 gene (>50 copies/cell) in all of the MCF-7 cells analyzed (Fig. 1A). This finding is consistent with previously identified amplification of the 17q23 chromosomal region in MCF-7 cells. The D283Med (D283) medulloblastoma cell line, with i17q, displayed low-level amplification of the WIP1 locus with four to nine copies in each interphase spread examined (Fig. 1B). In contrast, the Daoy medulloblastoma cell line with trisomy 17 displayed an average of three copies of WIP1 (Fig. 1C).
Fig. 1WIP1 copy number is increased in medulloblastomas with gain of chromosome 17q. High power micrographs of interphase nuclei of cell lines and primary human medulloblastoma cells hybridized to fluorescently-labeled WIP1 BAC (green) and centromeric chromosome 17 probes (red): (A) MCF-7 nucleus showing numerous WIP1 amplicons; (B) D283Med (D283) nucleus exhibiting low-level WIP1 amplification; (C) Daoy nucleus showing gain of 17; (D–L) interphase nuclei of medulloblastoma cells from nine patients, illustrating examples of (D–H) diploid and near-diploid nuclei, and (I–L) those with low-level amplification of WIP1
Among the 71 tumors analyzed by CGH, 11 primary medulloblastomas were examined for WIP1 amplification by FISH [24]. Six tumors displayed i17q, two showed trisomy 17, one had loss of 17p12-p13 with gain of 17p11.2-q25, and two had no change in chromosome 17 (Table 1). We detected low-level amplification of the WIP1 locus in 7 of 11 primary medulloblastoma specimens, which also had gain of 17q, including four with i17q by G-banding.
Table 1WIP1 amplification is associated with gain of chromosome 17q or i17q in primary medulloblastoma specimensSpecimenCopy number (FISH)Amplification*CNA (CGH)CEP 17WIP1WIP1Chr 17 statusD2832–44–9Yi17qDaoy43N+17MCF-74>50Y**MB1326–7Y−17p12-p13, +17p11.2-q25MB12–45–8Yi17qMB32–43–7Yi17qMB142–42–7Y+17MB152–42–6Y+17MB526Yi17qMB23–46–11Yi17qMB624Ni17qMB2822NN/CMB2722NN/CMB42–32–3Ni17qFISH analysis for WIP1 and chromosome 17 centromere reveals low-level amplification of WIP1 in 7 of 11 primary medulloblastoma samples. WIP1 copy gain is significantly associated with gain of 17q (P = 0.00057)*Amplification is defined as >4 copies as determined by FISH**Reportedly amplified, but not determined in the present studyAbbreviations: MB, medulloblastoma; +17, gain of chromosome 17; −17, loss of chromosome 17; N/C, no change in chromosome 17; CNA, copy number aberration; CEP 17, centromeric chromosome 17 probe
Four tumors were diploid or near-diploid overall (Fig. 1D–G), and seven displayed copy number gain of the WIP1 locus (Fig. 1H–L). Our previously reported CGH analysis of 71 medulloblastoma revealed that 46.8% had gain of 17q, including 30% with i17q and 2.6% with amplifications involving 17q, consistent with previous reports [4]. Comparing CGH results with our FISH analysis reveals that WIP1 DNA copy number correlates with gain of 17q (including i17q) by CGH (P < 0.001, Student’s t-test). All the tumors with copy number gain of WIP1 by FISH also had gain of the WIP1 locus on chromosome 17q22-q23 by CGH. WIP1 amplification in medulloblastoma was low-level compared to the high-level amplification observed in MCF-7 cells. These cytogenetic results indicate that low-level amplification of the WIP1 locus are prevalent in a significant proportion of medulloblastoma.
Medulloblastomas with gain of 17q, including i17q, overexpress WIP1 RNA
Using qRT-RTPCR, we determined the expression of WIP1 RNA in cell lines and a larger series of primary medulloblastoma samples. Human fetal cerebellum specimens displayed 2.1-fold greater WIP1 expression (±0.2, SEM) compared to fetal whole brain. Human adult cerebellum expresses 4.0-fold more WIP1 RNA (±1.0) than fetal total brain. WIP1-amplified MCF-7 cells had 14-fold higher levels of WIP1 RNA (±2.6), relative to human fetal brain (Fig. 2A). Daoy cells expressed 0.23-fold WIP1 RNA (±0.054, SEM), compared to fetal brain (Fig. 2A). In contrast, i17q-positive D283 cells displayed 9.8-fold (±1.8) higher expression of WIP1 RNA than human fetal brain.
Fig. 2Medulloblastomas with gain of 17q express significantly higher WIP1 RNA levels. (A) WIP1 RNA expression, by qRT-RTPCR analysis, is significantly higher in the D283 cell line and in primary medulloblastoma specimens with gain of 17q or i17q (+17q/i17q MB, n = 16) by conventional karyotyping and CGH, compared to Daoy, and primary tumors without i17q or gain of 17q (non-17q gain MB, n = 17). For reference, expression in the WIP1-amplified MCF-7 breast cancer cell line is also shown. Columns, mean expression of at least three experiments; error bars, SEM Y-axis, RNA expression normalized to GAPDH expression and relative to human fetal brain. (B) Relative WIP1 RNA expression in individual medulloblastoma specimens (n = 33) assayed by qRT-RTPCR, and listed with their associated chromosome 17 status. (C) Relative WIP1 RNA expression in medulloblastomas with WIP1 amplification (open diamond) is significantly higher than non-amplified tumors (open circle). Abbreviations: ALL MB, mean (±SEM) of all primary human tumors; i17q, isochromosome 17q; +17, gain of chromosome 17; −17, loss of chromosome 17; NC, no change in chromosome 17
We surveyed 33 primary medulloblastoma specimens for WIP1 expression and determined that RNA levels were significantly higher in 16 tumors with 17q gain or i17q (Fig. 2, Table 2). Of the primary medulloblastomas examined by FISH, WIP1-amplified tumors (n = 7) displayed significantly higher RNA levels than non-amplified tumors (n = 4) (27.7-fold (±1.6) vs. 14.1-fold (±4.5) more WIP1 RNA than fetal control; P = 0.050) (Fig. 2C, Table 2). Among the larger set of analyzed specimens, primary medulloblastoma samples displayed 13.0-fold higher levels of WIP1 RNA (±2.1) than human fetal brain (Fig. 2A–B, Table 2). Analyzed separately, tumors with gain of 17q, including i17q, (n = 16) expressed 20.3-fold more WIP1 RNA (±2.1), compared to those without gain of 17q or i17q (n = 17) with only 5.9-fold more WIP1 RNA (±1.0), relative to human fetal brain (Fig. 2A–B, Table 2). In other words, those tumors with gain of 17q, including i17q, expressed 3.4-fold more WIP1 RNA (±0.86) than those without such cytogenetic lesions. The association between WIP1 RNA expression and the presence or absence of 17q gain (including i17q) was statistically significant (P < 0.00001) (Table 2).
Table 2WIP1 overexpression is associated with gain of chromosome 17q or i17q in primary medulloblastoma specimensTumors grouped by CNAnWIP1 RNAP value+17q MB328.30.02*i17q MB625.4<0.0001*+17q or i17q MB926.3<0.00001*Non-17q gain MB26.5–WIP1 gain/amplification727.70.05**Normal WIP1 copy number414.1–Tumors analyzed by FISH (subtotal)11+17q MB520.20.0533*i17q MB1120.3<0.0001*+17q or i17q MB1620.3<0.00001*Non-17q gain MB175.9–All tumors analyzed by CGH (total)3313.0–WIP1 amplification by FISH was associated with increased WIP1 RNA expression by qRT-RTPCR (P = 0.05). Relative WIP1 RNA level was significantly associated with gain 17q, including i17q (P = 0.000001)*P value: Student t-test of tumors with specified CNA versus tumors without that CNA**P value: Student t-test of WIP1-ampllified versus non-amplified tumorsAbbreviations: MB, medulloblastoma; +17, gain of chromosome 17; −17, loss of chromosome 17; CNA, copy number aberration
Medulloblastomas with gain of 17q overexpress WIP1 protein
A subset of representative tumors with sufficient tissue was analyzed for WIP1 protein expression. Western immunoblot analysis confirmed significant protein expression in lysates from medulloblastoma cell lines and tumor samples, internally normalized to β-actin expression and relative to expression in the MCF-10A cell line (Fig. 3A–B). The medulloblastoma cell line, Daoy, with trisomy 17, expressed 3.1-fold more WIP1 protein (±0.5, SEM) than MCF-10A cells (Fig. 3A–B). The D283 cell line, with i17q, displayed 15.4-fold higher WIP1 levels (±2.0) than MCF-10A (Fig. 3A–B). Since UV irradiation activates p53 by phosphorylation at serine 15 and induces WIP1 expression [30], we used lysates prepared from irradiated D283 cells to provide control specimens with induced p53 and WIP1 proteins. In UV-irradiated D283 cells, p53 levels stabilized with increased phosphorylation at Ser15 (Fig. 3A). The WIP1-amplified breast cancer line, MCF-7, expressed 4.5-fold more WIP1 (±0.4) than MCF-10A cells (Fig. 3A–B).
Fig. 3Medulloblastomas express significant WIP1 protein. (A) Western blots of cell lines and four primary medulloblastoma specimens reveal significant WIP1 and p53 protein expression. UV irradiation (30 J/m2) of D283 increases WIP1 and p53 expression and induces p53 phosphorylation detected as phospho-p53 (Ser15). Breast carcinoma cell lines, WIP1-amplified MCF-7 and non-amplified MCF-10A are shown for reference. Associated chromosome 17 lesions are also labeled. (B) Western analysis of WIP1 protein expression among primary medulloblastoma specimens (n = 10), normalized to internal reference β-actin and relative to expression in the MCF-10A cell line. Abbreviations: all MB, mean (±SEM) of all primary human tumors (n = 10); i17q, isochromosome 17q; +17, gain of chromosome 17; −17, loss of chromosome 17; CNA, copy number aberration
Of the ten primary medulloblastoma specimens analyzed, five displayed gain of 17q or i17q, one had trisomy 17, another had loss of 17p, and three showed no change in chromosome 17 by CGH. Overall, mean WIP1 protein expression was 6.1-fold (±1.1) greater than expression in MCF-10A cells, internally normalized to β-actin (Fig. 3B). As a group, primary tumors exhibited even more WIP1 than the MCF-7 cell line, which is widely employed as an example of WIP1-amplification. Analyzed separately, primary medulloblastomas with 17q or i17q (n = 6) displayed 7.1-fold (±1.5) more WIP1 protein than MCF-10A, compared to non-17q gain tumors (n = 4) with only 4.6-fold (±1.3) levels (Fig. 3B). We also analyzed primary medulloblastoma lysates for expression of total p53 protein and p53 phosphorylation at serine 15 (Ser15) (Fig. 3). Total p53 and phospho-p53 (Ser15) were readily detectable on Western blots, and suggest functional significance. Overall, Western analysis indicated significant levels of WIP1 protein expression in primary tumors, generally higher in those with 17q gain or i17q, which might be responsible for antagonizing p53 activation.
Genotoxic stress induces p53 phosphorylation and WIP1 expression in medulloblastoma cell lines
To determine the functional significance of WIP1 overexpression, we transiently transfected established medulloblastoma cell lines and assayed changes in cell cycle and apoptosis in response to altered levels of WIP1. The D283 and Daoy cell lines differ in their basal WIP1 expression and their TP53 status, providing cell systems for gain-of-function transfection studies. Genotoxic stress in the form of exposure to UV irradiation or the chemotherapeutic agent VP-16 stabilized p53 levels and induced phosphorylation of p53 in D283 and Daoy cells, respectively (Fig. 4A). We examined the effects of increased constitutive WIP1 by transient transfection with a flag-tagged expression plasmid. Flag-tagged WIP1 could be readily distinguished from endogenous WIP1 on Western blots and WIP1 transfection was accompanied by decreased phospho-p53 (Ser15) in treated D283 and Daoy cells that are wild-type and mutant for TP53, respectively (Fig. 4A).
Fig. 4WIP1 antagonizes p53 activation in medulloblastoma cell lines. (A) Western blots of D283 and Daoy medulloblastoma cell lines: Treatment with UV irradiation (UV, 30 J/m2) or etoposide (VP-16, 1 μM) stabilizes wild-type p53 protein and induces phosphorylation of p53 at Ser15. Transfection with the WIP1-flag expression plasmid detected as higher molecular weight band using anti-WIP1 or anti-flag antibodies. Overexpressed WIP1 reduces p53 phosphorylation at serine 15 (Ser15). (B) Increased TP53 and WIP1 RNA in D283 and Daoy cell lines transfected with TP53-GFP and WIP1-flag (WIP1) expression plasmids compared to the MaxGFP control plasmid, as measured by qRT-RTPCR. (C) Luciferase assays reveal that co-transfection of WIP1-flag (WIP1) partially abrogates p53-dependent transcription from the p21-luciferase reporter (relative to constitutively expressed control Renilla-luciferase reporter) in TP53-GFP transfected D283 and Daoy cells (error bars, SEM). (D) TUNEL assay and flow cytometric methods confirm VP-16-induced apoptosis in Daoy and D283 cell lines
Determination of RNA levels with qRT-RTPCR confirmed significant expression of TP53 and WIP1 by transfection with WIP1-flag and TP53-GFP expression plasmids (Fig. 4B). To confirm the transcriptional effects of WIP1- and TP53-expression plasmids, we co-transfected cell lines with the p53-responsive p21-luciferase reporter plasmid and an actin promoter-driven Renilla luciferase plasmid to control for transfection efficiency. Transfected WIP1 partially abrogated p53-dependent luminescence indicating anti-p53 activity in both cell lines in vitro. Presumably because of endogenous wild-type TP53, the relative response in D283 cells was less dramatic than in Daoy cells (Fig. 4C).
VP-16 and constitutively expressed p53 induce apoptosis
Expression from epitope-tagged plasmids enabled us to employ flow cytometric methods to analyze the cell cycle distribution of fluorescently immunolabeled WIP1- and p53-overexpressing transfectants. To stimulate genotoxic responses in vitro, we employed minimally toxic dose concentrations of etoposide (VP-16), a chemotherapeutic topoisomerase II-inhibitor used to treat medulloblastoma clinically. VP-16-induced changes in apoptosis were also determined by flow cytometric quantitation of cells in sub-G0/G1 fractions, which were confirmed by TUNEL analysis (Fig. 4D). Similar results were obtained with micromolar concentrations of another chemotherapeutic agent, cisplatin (data not shown).
When exposed to low concentrations of VP-16, the D283 medulloblastoma cell line underwent increased apoptosis indicated by an increased fraction of cells in sub-G0/G1 by flow cytometric analysis, from 4.5 to 17.2% (P = 0.009) (Fig. 5F). These results indicate that VP-16 induces apoptosis, as described for other cell types with wild-type p53.
Fig. 5WIP1 antagonizes p53-mediated apoptosis and arrest in medulloblastoma cell lines. Flow cytometric analysis of cell cycle distributions of: (A) untreated D283 controls, illustrating the cell populations with 2n DNA content in G0/G1 phase, 4n DNA content in G2/M phase, intermediate DNA content in S phase, and the sub-G0/G1 peak representing apoptotic nuclei; (B) WIP1-flag transfected D283, (C) p53-GFP transfected D283, (D) WIP1-flag and p53-GFP (WIP1 + p53) doubly transfected D283, and (E) etoposide (VP-16)-treated D283 (Y-axis, cell number; X-axis, DNA content). Histograms summarizing flow cytometric analyses of transfected cultures: (F) apoptotic D283 and (G) apoptotic Daoy in Sub-G0/G1, with plasmids and treatment with V-16, as indicated (Y-axis, %; X-axis, transfected plasmid(s) and culture conditions). The following annotation symbols refer to statistically significant differences in sub-G0/G1 cell populations, as determined by paired Student t-test: * D283 cells transfected with p53-GFP plasmid versus mock-transfected D283 cells (P = 0.033). ** D283 cells co-transfected with both p53-GFP and Flag-WIP1 plasmids versus D283 cells transfected with p53-GFP alone (P = 0.026). *** D283 cells transfected with p53-GFP plasmid and treated with VP-16 vs. VP-16-treated, mock-transfected D283 cells (P = 0.019). # D283 cells transfected with Flag-WIP1 plasmid versus untreated, max-GFP control-transfected D283 cells (P = 0.013). ## Daoy cells transfected with p53-GFP plasmid and treated with VP-16 vs. VP-16-treated, mock-transfected Daoy cells (P = 0.044)
Increasing p53 in D283 cells with a GFP-tagged p53 expression plasmid also increased apoptosis (16.9% in sub-G0/G1) compared to mock-transfected controls (4.5%, P = 0.033) (Fig. 5F). VP-16 treatment of p53-transfected D283 cells further increased the sub-G0/G1 population to 25.6% (P = 0.019 compared to mock-transfected VP-16-treated controls) (Fig. 5F). These results suggest that additional constitutive p53 expression in transfected D283 cells can overcome negative regulatory pathways including endogenous MDM2 and WIP1.
We also transfected the TP53-mutant Daoy medulloblastoma cell line. Like wild-typeTP53 D283 cells, control Daoy cells treated with VP-16 increased their apoptotic sub-G0/G1 population to 20.4 vs. 6% in untreated controls, suggesting that VP-16 induces apoptosis even in the absence of wild-type p53 (Fig. 5G). Constitutive expression of transfected wild-type p53 in TP53-mutant Daoy cells enhanced VP-16-induced apoptosis to 33.5% (sub-G0/G1), compared to mock-transfected, VP-16-treated controls (14.6%, P = 0.044) (Fig. 5G). These data suggest that transfected p53 expression partially rescues TP53 mutation in Daoy cells.
WIP1 overexpression limits VP-16- and p53-induced apoptosis
We examined the effects of constitutive WIP1 expression in wild-typeTP53 D283 cells. VP-16 exposure increased apoptosis to 18.5 from 5.7% in untreated WIP1-transfected D283 (Fig. 5F). Despite limited transfection efficiency, extra WIP1 expression appeared to antagonize p53 activity even in the absence of genotoxic stress. When D283 cells were co-transfected to constitutively overexpress both WIP1 and TP53, they displayed fewer cells in sub-G0/G1 than p53-transfectants (10 vs. 16.9%, respectively; P = 0.026) (Fig. 5F). These data suggest that additional WIP1 limits exogenous p53-mediated apoptosis. In double WIP1- or TP53-transfected D283 cells, VP-16 increased apoptosis (15%) (Fig. 5F). Compared to VP-16-treated p53-transfected D283, double transfectants displayed a trend toward less apoptosis and fewer cells in G0/G1 phase (Fig. 5F). These data are consistent with anti-p53-induced apoptosis, presumably due to WIP1 overexpression, despite genotoxic stimulation.
WIP1-transfected Daoy cells lacking wild-type p53 did not differ in their cell cycle profile from control transfectants under basal conditions, demonstrating the p53-dependence of WIP1 effects. However, VP-16-treated WIP1-transfectants underwent more apoptosis (23.6%) than untreated counterparts (4.1%) (Fig. 5G). These data suggest a minimal effect of constitutively expressed WIP1 in the absence of wild-type p53. Overexpressed WIP1 also limited p53-mediated apoptosis in doubly co-transfected Daoy cells. As in controls, VP-16 treatment of double WIP1/TP53 Daoy transfectants increased cells in sub-G0/G1 (21.8%) (Fig. 5G). However, VP-16-treated double WIP1- or TP53-transfectants displayed fewer apoptotic cells compared to VP-16-treated single p53-transfectants. Thus, excess WIP1 also appears to protect VP-16-treated Daoy cells from p53-induced arrest and apoptosis.
Discussion
Regulation of the responses of medulloblastoma to genotoxic therapies is not well understood. Because TP53 has been implicated but rarely mutated or deleted in medulloblastoma, we focused attention on the candidate oncogene, WIP1. Recently published studies employed genomic methods to associate WIP1 with gain of 17q23 in medulloblastomas. Mendrzyk et al. used array-CGH to identify gain of chromosomal region 17q23.2-qter, a locus containing WIP1, in 24 of 47 (51%) medulloblastomas [22]. WIP1 mRNA was moderately overexpressed with respect to normal cerebellum control in seven of nine tumors. Immunostaining of a large series of tissue micro-arrays revealed strong nuclear expression in 148 of 168 (88%) tumor samples. Ehrbrecht et al. used conventional and array-CGH to identify amplification of 17q23 in three (19%) tumors and copy number gain of 17q23 in another 3 out of 16 medulloblastomas [23]. In 3 of 11 (27%) tumors, WIP1 mRNA was expressed less than fivefold in excess of expression in normal cerebellum control. Using additional approaches, we have confirmed gain of the WIP1 locus in medulloblastomas and quantitated its relative overexpression in our larger series of tumors. These results raise the possibility that overexpressed WIP1 functions as an oncoprotein in medulloblastoma.
WIP1 is a p53-dependent gene induced by genotoxic stress [20, 31]. As a PP2C serine–threonine phosphatase family member, Wip1 can counteract the tumor suppressive activity of p53 through several distinct mechanisms. WIP1 dephosphorylates p53 that has been activated in response to genotoxic stress [21]. WIP1 also dephosphorylates and stabilizes MDM2, promoting p53 degradation (Xiongbin Lu, manuscript submitted). This provides another feedback loop that returns the cell to a homeostatic state following DNA repair. Wip1 can complement different oncogenes in their transformation of mouse embryo fibroblasts. Wip1 overexpression also phenocopies the anti-apoptotic effects of TP53 mutation in vitro and increases tumorgenicity in murine models in vivo [20, 32].
Amplification or overexpression of WIP1 in breast and ovarian cancers may phenocopy the loss of p53 function, thereby contributing to growth and resistance to genotoxic therapies [17, 18]. In breast carcinomas with amplified and overexpressed WIP1, the TP53 locus is rarely mutated, suggesting that WIP1 amplification inhibits p53 activity and reduces selection for p53 mutations during tumor progression [17]. Although WIP1 expression reportedly correlates with clinical outcomes in patients with breast and ovarian carcinomas [33, 34], conclusive multivariate analysis of additional factors (such as the concurrent deletion of a TP53 allele and loss of 17p seen with i17q) awaits future analysis of greater tumor numbers.
Analysis of our series of primary medulloblastomas revealed low-level amplification of WIP1 as well as increased expression of WIP1 mRNA transcript and protein, especially in tumors with 17q gain and i17q. The connection between WIP1 amplification and these common cytogenetic lesions suggests a selective advantage. Since a few tumors with i17q did not display WIP1 amplification, other factors probably contribute to the emergence of this characteristic cytogenetic lesion. Conversely, one or more tumors without 17q gain displayed WIP1 overexpression. Although extra WIP1 copy numbers may explain relative overexpression via gene dosage effects, regulation at the post-transcriptional and -translational levels may also affect WIP1 protein levels. Consistent with our FISH results, qRT-RTPCR and Western blot analyses demonstrated increased levels of WIP1 mRNA and protein; both significantly associated with 17q gain and i17q. The degree of WIP1 RNA overexpression relative to normal cerebellar tissue corresponded to earlier reports [22, 23]. The apparent discrepancy between WIP1 expression at the RNA and protein levels suggests that post-translational modifications also contribute to WIP1 overexpression. The positive and negative regulatory networks appear to involve p38MAPK, MDM2, CHK1, and other DNA damage response factors [21, 30, 33].
Of note, our analysis was limited to those tumor specimens with available fresh tissue for molecular studies and displays an unintended selection bias for mixed tumors with anaplastic features. However, none of our findings appeared to correlate with histologic subtype or clinical features. Having confirmed the gain of WIP1 copy numbers and quantitated its relative overexpression in our medulloblastoma series, we hypothesized that overexpressed WIP1 contributes to treatment resistance by interfering with p53 function in medulloblastoma.
Because the tumor specimens were obtained before genotoxic treatment, their WIP1 and p53 levels do not reflect their activity. Therefore, we employed established cell lines to analyze the effects of overexpression of WIP1. Despite their phenotypic differences, D283 and Daoy cells provide readily manipulable systems for comparing the cell cycle effects of WIP1 in the setting of wild-type versus mutant p53. Our results indicate that chemotherapy-induced genotoxic stress or p53 overexpression alone can induce apoptosis in D283 cells. The constitutive overexpression of p53 overcame endogenous negative regulation in D283 cells, and rescued TP53-mutation in VP-16-treated Daoy cells. These results are consistent with studies of other p53 wild-type cell types, including the neuroepithelial lineage, indicating that VP-16 induces apoptosis in a p53-related manner [35–37].
Medulloblastoma cell lines displayed predicted effects of WIP1 overexpression. In addition to high basal WIP1 levels in D283 cells, additional transfected WIP1 counteracted the apoptotic effects of endogenous p53. The anti-p53 and chemoprotective effects of overexpressed WIP1 were also evident upon VP-16 treatment. These results support the hypothesis that WIP1 overexpression modulates genotoxic responsiveness by negatively regulating p53 in medulloblastoma cells. WIP1 overexpression appears to antagonize p53 activation, and provides medulloblastoma cells the ability to resist arrest and apoptosis due to genotoxic stress.
The frequency of WIP1 amplification and the degree of overexpression in primary medulloblastoma strongly suggests the acquisition of selective advantages that may explain the prevalence of 17q gain and i17q. Based on evidence from medulloblastoma cell lines , we hypothesize that WIP1 regulates p53 function, which contributes to tumor growth and genotoxic treatment resistance. The recent development and characterization of agents that inhibit WIP1 raise the possibility of therapeutic intervention in those medulloblastomas with wild-type TP53 [38, 39].

medulloblastoma
p53
wip1/ppm1d
isochromosome 17q
apoptosis
fish


Homocysteine affects cardiomyocyte viability: concentration-dependent effects on reversible flip-flop, apoptosis and necrosis

Background Hyperhomocysteinaemia (HHC) is thought to be a risk factor for cardiovascular disease including heart failure. While numerous studies have analyzed the role of homocysteine (Hcy) in the vasculature, only a few studies investigated the role of Hcy in the heart. Therefore we have analyzed the effects of Hcy on isolated cardiomyocytes.
Introduction
Hyperhomocysteinaemia (HHC) reflects an impairment of the methionine–homocysteine metabolism. Normally homocysteine (Hcy) is converted to cysteine via transsulfuration or to methionine by addition of a methyl group donated by folate [1]. However, when these pathways are deranged because of genetic mutations of enzymes involved or relative deficiencies of folate, vitamin B6 or vitamin B12, the serum concentration of Hcy increases [2, 3].
In 1969 Mc Cully et al. reported elevated concentrations of Hcy in young patients with severe cardiovascular disease [4]. They hypothesized the existence of a pathogenic link between HHC and atherogenesis. Since then other studies have also suggested that HHC is an independent risk factor for cardiovascular complications [5–8]. It is now assumed that even mild HHC constitutes an independent risk factor for cardiovascular disease [9] with equal predictive strength as hypercholesterolemia and smoking [10]. Whether Hcy itself or a Hcy-related substance such as S-adenosyl-homocysteine (SAH) is the culprit, and what the exact pathogenic mechanism of the cardiovascular complications induced by HHC could be, remains to be elucidated. In addition, large-scale intervention trails using high-dose-B-vitamins to reduce plasma Hcy levels in cardiovascular patients have as yet not shown an overall clinical benefit [11–13]. This raises further questions about the mechanisms underlying the association between HHC and cardiovascular disease, and about the possible adverse effects of high-dose-B-vitamins as a means of reducing Hcy levels [14].
Studies on the effects of Hcy on the vascular wall, especially on endothelial and smooth muscle cells, indicate that increased Hcy leads to cellular stress and also to cell damage [15–17]. Hcy primes human neutrophils for an increased production of reactive oxygen species (ROS) and induces ROS production in endothelial cells [15, 18]. ROS production has also been found in the vascular wall of coronary arteries during HHC [19] and a role of NADPH oxidases in this process has been suggested.
From the Framingham Heart Study it is known that increased plasma homocysteine concentrations, next to their effect on the vasculature, also form an independent risk factor for the development of heart failure [20]. Several other studies have also shown a link between HHC and heart failure [21, 22]. However, so far no studies have addressed possible effects of Hcy on cardiomyocytes. Therefore, we performed the present study to analyze the effects of Hcy on isolated cardiomyocytes.
Materials and methods
Cell cultures
Rat cardiomyoblasts (H9c2 (2–1) cells; ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagles Medium (DMEM; BioWhittaker, Verviers, Belgium) supplemented with 10% heat-inactivated fetal calf serum (FCS; BioWhittaker), 2 mM l-glutamine (GIBCO, Paisley, UK), 100 IU/ml penicillin (Yamanouchi Europe BV, Leiderdorp, Netherlands) and 100 μg/ml streptomycin (Radiopharma-Fisiopharma, Palomonte, Italy), and grown at 37°C in a humidified 5% CO2/95% air atmosphere. Experiments were performed with cells grown to a confluency of 60–80%. Adult rat cardiomyocytes were isolated as described before [23].
Antibodies and chemicals
Monoclonal Antibody 48 against NOX2 IgG 1 (dilution 1:28) was obtained from Sanquin Research at CLB, Amsterdam, The Netherlands [24]. Cy3-labeled goat-anti-rabbit-Ig (1:75) (Alexa Fluor; Leiden, Netherlands) was used as a secondary antibody. Polyclonal α-Nitrotyrosine IgG (1:50) (Molecular Probes Inc, Eugene, OR, USA) was used as an antibody to measure nitrotyrosin residues which is an indicator for ROS production. In this case, Cy5-labeled goat-anti-rabbit-Ig (1:50) (Jackson Immuno Research, West Grove, PA, USA) was used as a secondary antibody. Isotype controls and PBS were used to determine aspecific binding. Monoclonal antibody 1501R against pan-actin (1:6000; Chemicon International, Inc, Temecula, CA, USA) was used as a loading control for the western blot analysis.
Cells were incubated with several concentrations (0.1 mM–2.7 mM) of d,l-homocysteine (Hcy) (Sigma, St. Louis, MO, USA) in growth medium for 24 h at 37°C in a humidified 5% CO2/95% air atmosphere.
Measuring Hcy concentration in growth medium
The H9c2 cells were incubated with different concentrations of d,l-Hcy. Several studies have suggested that only the l form is bioactive [25, 26]. Next to d,l-Hcy concentration we therefore also measured the l-form. d,l-Hcy was measured by HPLC using fluorescence detection [27]. Intra- and interassay coefficients of variation (CVs; std dev/average *100%) were 1.8 and 3.5%, respectively. l-Hcy was measured by the Abbott IMx fluorescence polarization immunoassay (IMx; Abbott Laboratories, Abbott Park, IL, USA). Intra- and interassay CVs were less than 2 and 4%, respectively. The concentration of Hcy in culture medium was measured before incubation (t = 0) and after 24 h (final timepoint for the rest of the experiments) of incubation with H9c2 cells to determine the amount of putative degradation and/or uptake of the Hcy during this incubation period.
Determination of intracellular S-Adenosylmethionine (SAM) and S-Adenosylhomocysteine (SAH)
Since previous studies suggested that an increase in SAH is the culprit in cellular damage due to elevated levels of Hcy, we also determined the intracellular concentration of SAM and SAH in H9c2 cells after 24 h of incubation with d,l-Hcy. Tandem mass spectrometry (MS/MS) was used for the determination of SAM/SAH concentrations as previously described [28].
Flow cytometry
Phosphatidylserine exposure was assessed with FITC-labeled human recombinant annexin-V (Bender Med Systems, Vienna, Austria), while propidium iodide (PI; Bender Med Systems) was used to determine the permeability of the cellular membrane. Experiments were performed in the presence or absence of Z-Asp-Glu-Val-Asp-fluoromethylketone (Z-VAD FMK, 25 μM, Alexis Biochemicals, Lausen, Switzerland), a general caspase inhibitor, to differentiate between reversible flip-flop of plasma membrane phospholipids, apoptosis and necrosis [23]. After treatment with Hcy, the cells were trypsinized and centrifuged at 400g for 5 min at room temperature. Cells were then washed with serum-free DMEM, and resuspended in serum free DMEM containing annexin V (1:40) for 30 min in the dark at 37°C in a humidified 5% CO2/95% air atmosphere. Cells were subsequently washed and resuspended in serum free DMEM containing PI (1:40). Cells were measured with a FACSCalibur (Becton Dickinson, San Jose, CA, USA). Results were analyzed by Cell Quest Pro software (Becton Dickinson).
Detection of caspase-3 activity
Cells were grown in a 96−wells plate (20,000 cells/well). After treatment with Hcy and/or Z-VAD FMK, cells were lysed and incubated with DEVD-rhodamine 110 substrate (Roche, Mannheim, Germany) for 1 h at 37°C. Subsequently the amount of free rhodamine was determined at a microplate fluorescence reader (TECAN spectrafluor, Switzerland). The developed fluorochrome was proportional to the concentration of activated caspase-3 and could be quantified by a calibration curve of diluted free rhodamine. Each condition was measured in triplo per measurement (total of three measurements).
Immunofluoresence microscopy
To measure the expression of NOX2 and the putative formation of nitrotyrosin, cells were incubated with or without Hcy for 24 h in the 4-well chamber slides (Nalge Nunc International, Naperville, IL, USA). Cells were washed with PBS and fixated with 4% formaldehyde for 10 min at 37°C. Cells were subsequently washed with PBS, permeabilized with acetone–methanol (70%–30%) for 10 min at −20°C, and then washed again with PBS/Tween-20 (0.05% (v/v) Tween-20 in PBS). Subsequently cells were incubated with primary antibodies against NOX2 and nitrotyrosin for 60 min at room temperature followed by incubation overnight at 4°C. PBS and isotype controls were also tested to determine nonspecific binding of the antibodies and background signal. The following day the cells were washed with PBS/Tween and incubated with the secondary antibodies for 30 min at room temperature. After subsequent washes in PBS/Tween and PBS, the slides were covered in mounting medium containing DAPI (Vector Laboratories Inc, Burlingame, CA, USA) to visualize nuclei. Thereafter the slides were covered with coverslips.
Subsequently, cells were analyzed by means of a 3I MarianasTM digital imaging microscopy workstation (Zeiss Axiovert 200 M inverted microscope; Carl Zeiss, Sliedrecht, Netherlands), equipped with a nanostepper motor (Z-axis increaments; 10 nm) and a cooled CCD camera (Cooke Sensicam, 1280 × 1024 pixels; Cooke Co, Tonawanda, NY, USA). Visualization of NOX2 and nitrotyrosine was performed with a 40× air lens. The microscope, camera and data viewing process were controlled by SlideBookTM software (version 4.0.8.1; Intelligent Imaging Innovations, Denver, CO, USA).
Live cell analysis of ΔΨm
To visualize mitochondrial membrane potential (ΔΨm) via Life Cell Imaging, cells were loaded for 30 min at 37°C with 0.5 μmol/l of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Cell Technology Inc, Minneapolis, USA). In non-apoptotic cells, JC-1 exists as a monomer and is visible on the green channel (FITC). In healthy cells these monomers accumulate as aggregates in the mitochondria due to existing ΔΨm. These aggregates are visible on the red channel (CY3) when viewed with a fluorescence microscope. Cells were washed with PBS before viewing and subsequently analyzed by use of a 3I MarianasTM digital imaging microscopy workstation as described above.
Western blot analysis
After treatment H9c2 cells were harvested into modified ELB lysis buffer (250 mM NaCl, 0.1% Nonidet P-40, 50 mM HEPES pH 7.0, 5 mM EDTA, 0.5 mM DTT) wherein protease inhibitor cocktail (PIC, 1:40; Sigma) was added, and the cell suspension was mixed rigorously and then incubated for 30 min on ice. After determination of the protein concentration of the samples with the BCA protein assay kit (Pierce, Rockford, IL, USA), reducing sample loading buffer (0.25 M TRIS pH 6.8, sodium dodecyl sulfate (SDS), glycerol, 2-mercaptoethanol, bromophenol blue) was added and the samples were mixed and heated at 95°C for 10 min. A total of 50 μg protein of each sample was then subjected to SDS-PAGE, transferred to nitrocellulose membranes and analyzed for NOX2 expression with monoclonal antibody 48 (1:250 dilution) and for pan-actin with monoclonal antibody 1501R as a loading control (1:6000), followed by horseradish–peroxidase–conjugated rabbit–anti-mouse immunoglobulins (RαM-HRP; 1:1000 dilution; DakoCytomation, Glostrup, Denmark). The blots were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences AB; Uppsala; Sweden). Staining was quantified with a charge-coupled device camera (Fuji Science Imaging Systems; Düsseldorf, Germany) in combination with AIDA Image Analyzer software (Isotopenmessgeräte; Staubenhardt, Germany).
ATP measurement via Luciferase–Luciferin assay
After treatment cells were collected by trypsinization and centrifugation at 400g for 5 min. Cells were then counted and equal amounts were taken per condition. After centrifugation for 2 min (400g) the supernatant was discarded. The pellet was resuspended in 150 μl of ice-cold perchloric acid (PCA) 0.4 M. Cells were left on ice for 30 min and then centrifuged again for 5 min (2000g) at 4°C. To the isolated supernatant 7.5 μl of K2CO3 5 M was added to neutralize pH. Samples were subsequently stored at −80°C until measuring. The amount of ATP in the samples was determined with a Luciferase–Luciferin assay (Biaffin GmbH & Co KG, Kassel, Germany) according to the manufacturers’ protocol. Samples were measured in a FluoroNunc MaxiSorp plate (Nalge Nunc International, Rochester, NY). Luminescence was measured by using a Tecan GENios Plus reader (Tecan Benelux, Mechelen, Belgium).
Statistics
Statistics were performed with the SPSS statistics program (windows version 9.0). To evaluate whether observed differences were significant, One Way ANOVA with post hoc Bonferroni tests were used. All values are expressed as mean ± standard error of the mean (SEM). A P value (two sided) of 0.05 or less was considered to be significant.
Results
Measurement of the concentration of d,l-homocysteine (d,l-Hcy), l-homocysteine (l-Hcy), S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH)
We tested the effects of d,l-Hcy at concentrations of 0.1 mM, 1.1 mM and 2.7 mM, in accordance with previous studies in isolated vascular cells [15, 17, 26, 29–31]. We quantified the exact concentration of d,l-Hcy in medium added to the cells, via HPLC. As depicted in Table 1, the concentration of d,l-Hcy was according to the concentration added to the growth medium.
Table 1Concentration of d,l-Hcy and l-Hcy in culture medium on t = 0, l-Hcy after 24 h incubation, and intra-cellular concentration of SAM/SAH after 24 h incubationmM Hcy addedmM Hcy measured[SAM] (nM) t = 24[SAH] (nM) t = 24[d,l-Hcy] [l-Hcy] t = 0[l-Hcy] t = 240.0 00.0007 (+/−0.00009)0.002 (+/− 0.00004)464.9 (+/− 36.6)6.3 (+/− 0.7)0.1 0.14 (+/− 0.03)0.06 (+/− 0.01) 0.06 (+/− 0.01)468.3 (+/− 77.2)7.0 (+/− 2.9)1.1 1.08 (+/− 0.04)0.46 (+/− 0.03)0.32 (+/− 0.01)*319.6 (+/− 56.2)16.9 (+/− 6.8)2.7 2.73 (+/− 0.06)1.18 (+/− 0.05)0.84 (+/− 0.03)*204.4 (+/− 74.5)**164.8 (+/− 34.6)***All mediators were measured in n = 3. A significant decrease of l-Homocysteine (l-Hcy) can be seen at 1.1 mM and 2.7 mM after 24 h (* P < 0.001). At 2.7 mM a significant decrease of S-adenosylmethionine (SAM,** P = 0.006); but an increase in S-adenosylhomocysteine (SAH, *** P < 0.001) was detected, compared to all other concentrations
Since previous studies have shown that only the l form of Hcy is bioactive [25, 26], we also determined the l-Hcy concentration in medium by IMx. We found that 42.7%, 42.5% and 43.3% of the total d,l-Hcy amount 0.1 mM, 1.1 mM and 2.7 mM, respectively, in fact was l-Hcy at t = 0. Furthermore, we determined the concentrations of l-Hcy at 24 h, the final time point of incubation, and found a significant decrease in the concentrations of l-Hcy, namely for 1.1 mM a significant decrease of 0.14 mM l-Hcy; +/−0.011 (P < 0.001) and for 2.7 mM a significant decrease of 0.34 mM l-Hcy; +/−0.0301 (P < 0.001).
It has been well documented that increased levels of SAH inhibit several important methylation reactions [32–34]. Therefore we also determined the intracellular concentrations of both SAH and SAM, being one of the primary methyl donors (Table 1). Measuring intracellular concentrations of SAM and SAH in H9c2 cells incubated with and without d,l-Hcy showed a significant depletion of SAM at 2.7 mM d,l-Hcy compared to untreated cells and 0.1 mM d,l-Hcy treated cells (P = 0.006; n = 3). While the concentration of SAH increased significantly at this same concentration compared to all other conditions (P < 0.001; n = 3). Therefore we also examined the effects of the higher, non-physiological concentrations of Hcy.
Effect of Hcy on cell viability
We analyzed the effect of Hcy on H9c2 cell viability by measuring annexin V and/or PI positivity using flow-cytometry. Incubation with 0.1 mM d,l-Hcy had no effect on single-annexin-V-positivity (Fig. 1A), double-annexin-V, PI positivity (Fig. 1B) or single-PI positivity (not shown, approximately 3% for each condition). In contrast, incubation with 1.1 mM d,l-Hcy resulted in a significant increase in single- annexin-V-positive cells (P < 0.002) (Fig. 1A). This increase in single-annexin-V-positive cells was only partly, but not significantly inhibited by Z-VAD FMK (31.6% decrease).1.1 mM d,l-Hcy had no significant effect on the percentage of double-annexin-V/PI-positive cells (Fig. 1B) or on the percentage of single-PI-positive cells (not shown). Incubation with 2.7 mM d,l-Hcy, however, resulted in a significant increase of single-annexin-V positive cells (P < 0.001) and double-annexin-V/PI-positive cells (P < 0.044) that could not be inhibited significantly by Z-VAD FMK. Again we did not see any significant increase in single-PI-positive cells (not shown). Thus, depending on the concentration of Hcy, either reversibly flip-flopped cardiomyocytes and/or late apoptotic/necrotic cells can be found.
Fig. 1Effect of Hcy on cell-viability via flow-cytometry: Flow cytometry analysis of H9c2 cells incubated with different concentrations of d,l-Hcy during 24 h, with or without 25 μM Z-VAD FMK, a pan caspase inhibitor. A total of 10,000 cells were measured (n = 3). (A) Percentage of single annexin V positive, PI-negative labeled cells, which is a marker of flip flop of the plasma membrane phospholipids. (B) Percentage of double-annexin-V/PI-positive cells depicting late apoptotic and/or necrotic cells. Data represent mean percentages and standard error of the mean (SEM)
Caspase-3 activity
To determine whether the population of double-annexin-V/PI-positive cells consists of late apoptotic cells or necrotic cells, we performed an assay of caspase-3 activity. Incubation with 0.1 mM and 1.1 mM d,l-Hcy did result in a small, but not significant increase in caspase-3 activity that was completely inhibited when Z-VAD FMK was added. Incubation with 2.7 mM d,l-Hcy for 24 h, however, induced a significant increase of caspase-3 activity (P < 0.001) that was also completely inhibited with Z-VAD FMK (Fig. 2). Caspase-3 activity measured at earlier time points (6 and 16 h) of Hcy incubation was not significantly increased (results not shown). Thus, significant apoptosis induction of H9c2 cells was only present at 2.7 mM d,l-Hcy.
Fig. 2Effect of Hcy on caspase-3 activity: H9c2 cells were incubated during 24 h with different concentrations of d,l-Hcy in the presence and absence of Z-VAD FMK (n = 9). Caspase-3 activity was measured as indicated under Methods. Data represent mean and SEM
Effect of Hcy on NOX2 expression and nitrosylation
In a previous study we have found that ischemia of cardiomyocytes induced nuclear NOX2 expression coinciding with nuclear nitrotyrosine residues, resulting in subsequent apoptosis of these cells [35]. Given the pro-apoptotic effect of 2.7 mM d,l-Hcy, we analyzed by Western blotting the effect of Hcy on NOX2 expression. We found that NOX2 was constitutively present in whole cell lysates of H9c2 cells (Fig. 3). NOX2 expression was slightly increased at 0.1 mM d,l-Hcy and increased even more at concentrations of 1.1 mM and 2.7 mM of d,l-Hcy. It is noteworthy that although we found this increase in NOX2 expression, we were not able to detect apoptosis at the lower concentrations of Hcy. Therefore we subsequently analyzed the intracellular localization of NOX2 with digital imaging. In the cytosol, we found diffuse positivity for NOX2 at all concentrations of Hcy (data not shown). Incubation with 0.1 mM and 1.1 mM, but especially with 2.7 mM d,l-Hcy resulted in a significant increase in NOX2 expression in the nucleus (P < 0.001; P = 0.047; P < 0.001, respectively) (Fig. 4A). These results appear in contrast with the caspase-3 measurements. We therefore also analyzed the presence of nitrotyrosine residues as an indicator for ROS production. We found that neither 0.1 mM nor 1.1 mM d,l-Hcy resulted in significant nitrotyrosine formation in the nucleus. In contrast, 2.7 mM d,l-Hcy did induce a significant formation of nitrotyrosine residues in the nucleus (P < 0.001) (Fig. 4B). These results underline that not only nuclear NOX2 expression but also nuclear ROS production is necessary for the induction of apoptosis. It has to be noticed that especially at 2.7 mM d,l-Hcy a significant loss of approximately 50% of the cells was found (P < 0.001, results not shown). As these cells theoretically are predominantly double-annexin-V/PI or single-PI-positive, we conclude that the effect of incubation with 2.7 mM d,l-Hcy on the amount of double-annexin-V/PI or single-PI-positive cells (see Fig. 1) in all probability is underestimated.
Fig. 3Effect of Hcy on NOX2 expression: (A) Western Blot analysis for NOX2 expression of whole-cell lysates from H9c2 cells incubated with or without Hcy for 24 h. Data represent mean and SEM for three independent experiments. (B) Western Blot analysis of the same blots for pan-actin as a loading controlFig. 4Effect of Hcy on intracellular localization of NOX2 and nitrosylation in H9c2 cells: Analysis of the effect of different concentrations of D,L-Hcy at 24 h incubation on the nuclear localization of NOX2 expression (A) and nitrotyrosine residues (B) measured by means of digital imaging microscopy. The arbitrary units are defined as sum intensity per nucleus and depicted as % increase versus control where control was set to 0 (n = 6). Data represent mean and SEM
We next wanted to corroborate the findings of NOX2-mediated nitrotyrosine formation in adult rat cardiomyocytes. Because the Hcy concentrations analyzed in H9c2 cells were lethally toxic for adult rat cardiomyocytes, we used lower concentrations for these cells. Incubation with 0.07 mM d,l-Hcy induced significant NOX2 expression in the nucleus (P < 0.022; Fig. 5D) but no significant nitrotyrosine formation. Incubation with 0.1 mM d,l-Hcy led to significant nuclear NOX2 expression and presence of nitrotyrosine residues (P < 0.01; Fig. 5A–E). As with H9c2 cells, we found only little NOX2 positivity in the cytosol under all Hcy concentrations analyzed (arrow in Fig. 5A).
Fig. 5Effect of Hcy on intracellular localization of NOX2 and ROS production in adult rat cardiomyocytes: Digital Imaging microscopy pictures of adult rat cardiomyocytes incubated with 0.1 mM d,l-Hcy during 24 h. Red color (A) indicates NOX2 expression which is visible in the nucleus and in the cytosol (arrow). Green color (B) represents nitrotyrosine formation. The merge image (C) also shows blue DAPI staining for DNA which shows co-localization of NOX2 expression and nitrotyrosine formation in the nucleus. (D) Amount of nuclear NOX2 expression in adult rat cardiomyocyt in control, 0.07 mM and 0.1 mM d,l-Hcy samples (n = 3). (E) Amount of nitrotyrosine formation in adult rat cardiomyocyt in control, 0.07 mM and 0.1 mM d,l-Hcy sample (n = 3). The arbitrary units are defined as sum intensity per nucleus and depicted as % increase versus control where control was set to 0. Data represent mean and SEM
ATP depletion
An important determinant in the process of cell death is ATP, as it is for instance necessary to complete the process of apoptosis. We therefore measured ATP concentrations in H9c2 cells (Fig. 6). Incubation with 0.1 mM d,l-Hcy resulted in a significant increase in ATP concentration (P = 0.05). In contrast, incubation with 1.1 mM d,l-Hcy led to a decrease in ATP concentration that was not significant compared to control cells, but was significant compared to ATP concentration found in cells that were incubated with 0.1 mM d,l-Hcy (P = 0.024). Incubation with 2.7 mM d,l-Hcy induced an more pronounced decrease of ATP concentration that was significant also when compared to control cells (P = 0.036).
Fig. 6Effect of Hcy on ATP levels: Increase or decrease in ATP levels (as compared to control levels) in H9c2 cells after incubation with different concentrations of d,l-Hcy during 24 h. Data represent mean and SEM of four independent experiments
ΔΨm
To explain the contrasting effects of different Hcy concentrations on ATP concentrations, we analyzed the mitochondrial membrane potential (ΔΨm) with JC-1 staining in H9c2 cells. We found an increase in red signal (P = 0.006) when the H9c2 cells were incubated with 0.1 mM d,l-Hcy compared to control cells (Fig. 7A/B) which is indicative for more actively respiring mitochondria. ΔΨm after incubation with 1.1 mM and 2.7 mM d,l-Hcy did not differ significantly from that in control cells (Fig. 7A–E). A difference in the morphology of mitochondria was also visible. In control cells a heterozygous mitochondria population is visible consisting of round and elongated mitochondria. Incubation with 0.1 mM d,l-Hcy showed mostly elongated mitochondria, whereas incubation with 1.1 mM and 2.7 mM d,l-Hcy resulted in a more rounded morphology of the mitochondria, the so called thread-to-grain transition which is seen as an early marker for apoptosis [36].
Fig. 7Effect of Hcy on mitochondrial membrane potential: H9c2 cells were incubated with or without Hcy for 24 h and analyzed with digital imaging microscopy. Green indicates the JC-1 monomers. Red indicates JC-1 aggregates, which are formed in the mitochondria when a sufficiently high membrane potential is reached. (A) H9c2 cells incubated in growth medium alone. Normal heterozygous population of elongated and round mitochondria have a red fluorescence signal. (B) H9c2 cells incubated with 0.1 mM d,l-Hcy. An increase in actively respiring mitochondria (red) is visible, and a mostly elongated morphology can be seen. (C) 1.1 mM d,l-Hcy shows a similar amount of functioning mitochondria in the cells that did not receive Hcy; only the morphology of the mitochondria is mostly round, a phenomenon which is known as thread-to-grain transition. (D) H9c2 cells incubated with 2.7 mM d,l-Hcy. Some cells have no functioning mitochondria left, but overall there is no significant loss of red signal. The thread-to-grain transition of the mitochondrial reticulum is complete. (E) Analysis of sum intensity in Cy3 signal (red) per condition as an indicator for ΔΨm. Data represent mean and SEM (n = 3)
Discussion
In the present study we examined the effects of Hcy on cell viability of cardiomyocytes and found different effects, depending on Hcy concentration (Fig. 8).
Fig. 8Effects of different concentrations of Hcy on cardiomyocyte viability Scheme of the proposed events caused by Hcy, which ultimately lead to cell death: (A) control cells. (B) 0.1 mM d,l-Hcy results in increase of ΔΨm and ATP levels, and translocation of NOX2 to the nucleus. (C) 1.1 mM d,l-Hcy results in flip-flop of the plasma membrane (bended arrows in plasma membrane indicate flip-flop of phophatidyl serine to the outer leaflet of the membrane), coinciding with localization of NOX2 in the nucleus. (D) 2.7 mM d,l-Hcy next to flip-flop and NOX2 localization in the nucleus, nuclear ROS production is found. ATP is then decreased, as well as ΔΨm. Caspase-3 is activated and PI can enter the cell indicating cell death
The lower concentration of 0.1 mM Hcy resulted in mitochondrial hyperpolarization, an increase of ATP and translocation of NOX2 to the nucleus but without local nitrotyrosin formation. Mitochondrial hyperpolarization and an increase in ATP concentration have been described as first signs of mitochondrial disturbance on the pathway to apoptosis [36, 37]. On the other hand, they have also been described as part of a cell-protective mechanism [38]. Therefore, the effect of this lower Hcy concentration may be a cellular state characterized by early activity of both pro- and anti-apoptotic pathways, which does not necessarily result in cell death.
In contrast, the intermediate concentration of 1.1 mM Hcy led to rounding of mitochondria, a slight, non significant decrease of ΔΨm with a visible thread-to-grain transition of the mitochondrial reticulum, a slight decrease in ATP concentrations, a translocation of NOX2 to the nucleus, again without presence of nitrotyrosin residues, and a flip-flop of the cell membrane phospholipids as indicated by single annexin v positivity. We did not find caspase-3 activation at this concentration. Although annexin V positivity of cells has generally been considered as a marker for apoptosis, in a previous study however, we have demonstrated that this is not absolute as such and that membrane phospholipid flip-flop can be reversible [23], which is supported in this study by the observation that complete inhibition of caspases by Z-VAD-FMK did not affect this phenotype. The caspase-dependent pathway of apoptosis was, therefore, not yet activated at this concentration of Hcy, probably related to the lack of nuclear nitrotyrosin residues.
The highest concentration of 2.7 mM Hcy entailed a completion of the thread-to-grain transition of the mitochondrial reticulum, a further decrease of ATP, translocation of NOX2 to the nucleus and nuclear nitrotyrosin formation, caspase-3 activation, and also a clear increase in both single-annexin-V and annexin-V/PI-positive cells. These changes represent a cellular phenotype irreversibly committed to cell death, in which nuclear ROS production apparently plays a deciding role as indicated by the presence of nitrotyrosin residues. Whether cell death then follows the process of apoptosis or necrosis depends on the remaining concentration of ATP. Apoptosis is energy consuming, and therefore depends on minimal residual ATP concentrations, whereas necrosis is the default route in the absence of ATP [39].
Induction of apoptosis generally follows one of two pathways. The first pathway, the death receptor or extrinsic pathway, is activated by ligand binding of cell-surface-expressed death receptors. The second pathway, the mitochondrial or intrinsic pathway, is promoted by physical or chemical stress agents [40]. Intracellular accumulation of Hcy and its metabolites constitutes considerable intracellular stress [15, 17, 41]. In line with this notion, several studies in endothelial cells have suggested that Hcy-induced apoptosis follows the mitochondrial pathway [42,43]. Our study in cardiomyocytes has shown that ΔΨm is modified in a concentration-dependent manner, and thereby supports the concept that Hcy-induced apoptosis mainly follows the mitochondrial pathway, also in cardiomyocytes. However, in addition we found that translocation of NOX2 to the nucleus coinciding with the presence of nitrotyrosin residues indicative for ROS production is even more important in the effector phase of the process of apoptosis induction in cardiomyocytes.
Since we only found a significant decrease in L-Hcy at the highest concentrations, but not with 0.1 mM d,l-Hcy we also determined SAH concentrations. Several studies provided further evidence that SAH could be the culprit in Hcy-mediated damage since it is a potent inhibitor of various methylation reactions [32–34]. In this study we found that only the concentration of 2.7 mM d,l-Hcy resulted in a significant increase in SAH, which was the concentration that resulted in cell death of cardiomyocytes. Therefore our study underlines that SAH is an important mediator in the induction of cell death of cardiomyocytes by Hcy.
A limitation of the present study is the use of relatively high concentrations of Hcy. However, it should be noted that cells in culture usually up-regulate their Hcy-metabolizing enzymes, in particular cystathionine beta-synthase (CBS) [44]. Furthermore short-term exposure to high Hcy concentrations may reflect a prolonged exposure to moderately elevated Hcy concentrations as occur life-long in patients. Therefore, our data may provide, at least in part, an explanation why HHC contributes to heart failure.
Conclusion
Hcy is increasingly recognized as an independent cardiovascular risk factor [5–8] and an extensive literature exists that links Hcy-induced changes in endothelial [15, 26, 45–48] and smooth muscle cells [16, 17, 49–52] to atherosclerotic disease. The Framingham Heart Study also showed that increased plasma Hcy levels are a risk factor for the development of heart failure [20]. To our knowledge, no reports have yet been published on the effect of Hcy on cardiomyocytes specifically. Our study now clearly indicates that Hcy, and possible SAH, indeed affects also cardiomyocytes, primarily by inducing apoptosis/necrosis in a concentration-dependent manner, in which nuclear ROS production, related to nuclear NOX2 expression, is crucial.

cardiomyocytes
apoptosis
hyperhomocysteinaemia
heart failure
oxidant stress


Improving Web Searches: Case Study of Quit-Smoking Web Sites for Teenagers

Background The Web has become an important and influential source of health information. With the vast number of Web sites on the Internet, users often resort to popular search sites when searching for information. However, little is known about the characteristics of Web sites returned by simple Web searches for information about smoking cessation for teenagers.
Introduction
The World Wide Web, with over 3 million public Web sites and over 1.4 billion Web pages [1], has become an important and influential source of health information [2]. In September 2002, there were an estimated 605 million people online worldwide [3]. In the United States, 90% (48 million) of the children and adolescents between the ages of 5 and 17 use computers, and 75% of the 14 to 17 year olds use the Internet [4]. With the vast amount and dynamic nature of information on the World Wide Web, it is not surprising to find that over 75% of those online use search sites to navigate the Web [5]. However, the amount of results returned from a search is often overwhelming. For example, 115000 results were found with the search terms "teen quit smoking" in Google.
Of the several thousand search sites or directories [6], only a few are of high popularity as indicated by their audience reach and time spent on them [7]. Although Google will provide up to a thousand results from a query, few users are likely to examine them all. In an observational study on 16 adult subjects, only 9 participants ever looked beyond the first search pages and only 5 of them ever clicked a link on those pages [8]. A survey done in 2002 on 1403 e-mail participants showed that only 23% of the users went beyond the second page [9]. Another pilot study of 12 teenagers found they looked past the fourth page of results less than 5% of the time [10]. Thus, position ranking in Web-search results, especially on the first few pages, is an important determinant of information accessibility by users.
Several studies have reported substantial variability in health-related Web-site content [11- 14]. While guidelines for evaluating the quality of health information on the Web are available [15- 19], the correlation between these guidelines and accuracy of health information is debated [20- 22]. Position ranking in search results was not associated with content quality [23]. Using the search term "breast cancer," Meric et al [24] reported that popularity of Web sites was associated with type rather than quality of content. In a sample of 75 Web sites that provided information on urinary incontinence, the Internet popularity indexes—as measured by the number of links to the main incontinence page of each Web site and by the number of links to all pages of each Web site divided by the number of pages of the site—were not correlated with a quality score based on Silberg et al [16] and the HONcode principles [25].
The aim of this study was: (a) to identify the characteristics of Web sites with information on smoking cessation for teenagers that ranked in the top 30 positions in a typical Web search on popular search sites and (b) to evaluate the association between those characteristics and the position ranking for sites that are of direct relevance to smoking cessation for teenagers. The findings are relevant for improving consumer access to health information.
Methods
This study was carried out from May 2003 through June 2003. Web sites with information on smoking cessation for teenagers were identified with 4 popular search sites using a specific search term. The characteristics of the identified sites were collected with a Web-site characteristic checklist; 2 raters evaluated each Web site independently (details below).
Search Protocol
Four popular search sites (Table 1) were used in this study. Users spend over 5 million search hours per month at each site. A search hour equals the number of visitors to a site multiplied by the average number of hours each visitor is estimated to have spent at the site.
Table 1
Popular search sites in the United States*
Search Site
URL
Total Search Hours (Millions of Hours) in January 2003
MainUnderlying SearchEngine
Google
www.google.com
18.7
Google
AOL
search.aol.com
15.5
Google
Yahoo!
www.yahoo.com
7.1
Google and Overture (for paid listings)
MSN
search.msn.com
5.4
LookSmart, Inktomi, Microsoft proprietary editor, and Overture (for paid listings)
* Source: Search Engine Watch [26].
The search term on smoking cessation for teenagers was selected based on information from the Overture Search Term Suggestion Tool [27] and the 7search Keyword Suggestion Tool [28]. These sites provide a count of the search terms that were submitted to their search engines. Overture provides their search results to various popular search sites including Yahoo, MSN, AltaVista, Lycos, HotBot, and AllTheWeb [29]. For example, in April 2003 there were 40036 searches submitted to Overture with "quit smoking," 27812 with "stop smoking," and 9001 with "smoking cessation." Various other combinations of "teen," "youth," "adolescent," "quit smoking," "stop smoking," and "smoking cessation" were compared. Based on the frequency of searches performed on the Web as recorded by the Overture database, the search terms "teen quit smoking" were submitted to the 4 search sites to locate sites with information on smoking cessation for teenagers.
To mimic the search behavior of Web users, only the top 30 search results were included in the study. Sites ranking below the top 30 results are likely to be found only by more-persistent searchers [30]. Thirty results are equivalent to 3 pages (2 clicks) of the default number of results per page in Google and AOL, 2 such pages (1 click) in MSN, and one and a half such pages (one click) in Yahoo. The results from the 4 search sites were combined into one list to provide an overall picture of the search activity on the Web. The sites were reranked by first grouping the sites into 4 groups by the number of search sites that included them (1 to 4 search sites) and then by the position ranking provided by the search results within each group. The top 30 reranked sites formed the sample for the analysis.
Since the rankings of Web sites within search-site results change frequently, the search results were captured in spreadsheet format using the Google API Search Tool [31]. The Web pages of sites identified by search results were captured using Offline Explorer software [32] to facilitate the recall of the exact page content when necessary and to provide consistency for the 2 raters.
Table 2
Site characteristics and correlation with search ranking for 14 sites relevant to teenagers who are seeking information on smoking cessation
Site Characteristic
Inter-Rater Reliability (Kappa)
Characteristics
Number (%)
Kendall Rank Correlation, Tau (P value)
Essential
Search feature in the site
0.86
Present
8 (57)
0.15 (.52)
Absent
6 (43)
Site navigation system on page
0.76
Present
11 (79)
-0.16 (.42)
Absent
3 (21)
Privacy statement
0.57
Present
8 (57)
0.18 (.41)
Absent
6 (43)
Disclaimer
0.69
Present
9 (64)
0.11 (.70)
Absent
5 (36)
Readability grade level
NA*
< 8.0
5 (36)
-0.39 (.05)
≥ 8.0
9 (64)
Broken links on page
NA
Present
7 (50)
0.08 (.75)
Absent
7 (50)
Enhancement
Indication of sponsorship
0.19
Present
11 (79)
0.09 (.66)
Absent
3 (21)
Pop-up advertisements or banner advertisements
0.59
Present
4 (29)
0.18 (.17)
Absent
10 (71)
Contact e-mail address
0.51
Present
10 (71)
-0.46 (.01)
Absent
4 (29)
Phone number or mailing address
1.00
Present
4 (29)
0 (1.00)
Absent
10 (71)
Content on cessation method: behavioral approach
0.43
Present
11 (79)
0.31 (.10)
Absent
3 (21)
Content on cessation method: medication approach
0.84
Present
10 (71)
-0.43 (.02)
Absent
4 (29)
Content on cessation method: alternative approach
0.51
Present
5 (36)
-0.42 (.02)
Absent
9 (64)
Annotated external hyperlinks
0.72
Present
5 (36)
-0.39 (.04)
Absent
9 (64)
Interactive component (quiz, game, or bulletin board)
0.53
Present
8 (57)
-0.18 (.44)
Absent
6 (43)
Material in video or audio format
1.00
Present
1 (7)
-0.20 (.31)
Absent
13 (93)
Technical
Page size (kilobyte)
NA
< 35
6 (43)
-0.39 (.04)
≥ 35
8 (57)
Meta description tag
NA
Present
8 (57)
-0.48 (.002)
Absent
6 (43)
Meta keywords tag
NA
Present
11 (79)
-0.31 (.13)
Absent
3 (21)
Persistent cookies
NA
Present
3 (21)
-0.34 (.06)
Absent
11 (79)
Part of a larger Web site
0.72
Yes
6 (43)
0.03 (.90)
No
8 (57)
Link density (reverse links)
NA
1
6 (43)
-0.58 (.02)
2-100
4 (29)
> 100
4 (29)
* NA = Not applicable. Kappa values for these characteristics were not available because they were analyzed by the WebXact Watchfire Page Analysis [35], except for readability grade level which was evaluated by only 1 rater.
Checklist of Web-Site Characteristics
A checklist was uses to evaluate the characteristics of the Web sites (see Table 2 for checklist items). The readability was estimated by the Flesch-Kincaid grade-level score [33]. (The Flesch-Kincaid grade-level score rates text on a United States grade-school level. For example, a score of 8.0 means that an eighth grader can understand the document.) Sample passages from the Web pages with information pertaining to smoking cessation of the identified sites were pasted into Microsoft Word XP for Windows to obtain the score. The results were recorded in a spreadsheet and subsequently imported into SPSS [34] for analysis. The number of broken links, page size, presence of meta tags, and presence of persistent cookies were obtained from WebXact Watchfire Page Analysis [35]. (Meta tags are HTML [hypertext markup language] tags that provide information about the content of a Web page for indexing by search engines but do not affect how a Web page is displayed by a browser.) Link density was obtained by using a reverse-lookup query (link:siteURL, where siteURL is replaced by the Web site's URL) in Google. The link density of a site is the number of external sites that have a link to that site. A site with a higher link density is generally more likely to be found by visitors because they may find it through the external sites.
Statistical Analysis
Correlations between position ranking and the Web-site characteristics were calculated using the Kendall rank correlation. The value of the coefficient (tau) ranges from -1 to 1. A value of zero indicates no correlation, values near 1 indicate a strong direct correlation, and values near -1 indicate a strong inverse correlation. Interobserver reliability between the 2 raters was calculated using Kappa statistics on all variables except readability, link density, and those returned by WebXact Watchfire Page Analysis. We regarded P£ .05 as statistically significant.
Results
Of the top 30 sites identified by the 4 search sites using the search terms "teen quit smoking," only 14 were relevant to teenagers who are seeking information on smoking cessation. We also evaluated the search results from Google by using other similar search terms. The number of relevant sites ranged from 5 to 17 (Table 3). Although we used only 1 search site to illustrate the effect of search terms on the type of Web sites found, the result should be similar at other search sites.
Characteristics of the 14 Relevant Web Sites
The characteristics of the 14 sites are summarized in 3 categories (Table 2).
Essential-Characteristic Category
The essential-characteristic category contains those characteristics that contribute to user dissatisfaction if absent or inadequately provided. The presence of a privacy statement and disclaimer, although it appears not to be required for the functioning of a Web site, wasreported to be essential in a Web-user interface study [36].
The correlation between the 2 raters ranged from 1.00 for 2 characteristics (presence of phone number or mailing address and presence of material in video or audio format) to 0.19 for indication of sponsorship. The median correlation was 0.69 for the 15 characteristics evaluated by both raters.
In the essential category, 8 sites (57%) contained a site-search feature and 11 sites (79%) contained links for navigation in the site. However, 2 sites contained neither of the features. Over half of the sites contained either a privacy statement (57%) or a disclaimer (64%) but only a third of the sites contained both. About one-third of the sites have readability below eighth-grade school level and they ranked significantly higher (tau = -0.39, P= .05) than those that have readability above or equal to that level. The median grade level was 8.5. Half the sites contained one or more broken internal or external hyperlinks.
Enhancement-Characteristic Category
In the enhancement-characteristic category, 11 sites (79%) indicated their sponsorship. Apparently because most of the sites were sponsored by organizations, government bodies, or educational institutions, only 4 sites (29%) had either pop-up advertisements or in-page banner advertisements. E-mail address (71%) was the most-common contact information available while phone number or mailing address was present in 29% of the sites. Sites that ranked higher were significantly associated with the presence of e-mail address for contact (tau = -0.46, P= .01). Eleven sites (79%) had information on behavioral approach as a method of smoking cessation. Ten sites (71%) had information on a medication (nicotine replacement) approach, and 5 sites (36%) had information on alternative approaches such as acupuncture, hypnosis, laser therapy, and herbal cigarettes. Both the presence of medication (tau = -0.43, P= .02) and alternative approaches (tau = -0.42, P= .02) were significantly associated with a higher search ranking. Five sites provided annotated hyperlinks to external sites and their presence was significantly associated with a higher search ranking (tau = -0.39, P= .04). Eight sites contained interactive components such as quizzes, games, or bulletin boards. Only 1 site provided material in video or audio format.
Table 3
Type of Web sites found with different search terms using Google search site
Type of Web Site
Search Terms Used
teen quit smoking
teen stop smoking
teen smoking cessation
youth quit smoking
adolescent quit smoking
Site with information to help teenagers quit smoking
14
5
5
17
5
Page with hyperlinks to Web sites with information to help teenagers quit smoking
3
1
5
3
4
News or press release
4
5
3
3
5
Report of study results or proceedings from conferences
1
2
5
2
9
Recruitment of study subjects
2
1
1
0
0
Commercial site
3 (2 were redirects*)
4 (1 was a redirect)
0
0
2 (both redirects)
Site for teenagers but not on smoking
1
2
0
0
0
Resources on teenager smoking cessation for parents or health professionals
1
4
5
3
2
Health organizations or community centers
0
1
3
1
1
Page not found
1
4
2
1
0
Other
0
1 (alt.support.stop-smoking Usenet archive)
1 (porno-graphic Web site)
0
2 (mental health Web site)
* The visitor was automatically sent to a page other than the page listed in the search results (see Discussion for details).
Technical-Characteristic Category
In the technical-characteristic category, the largest file size of the landing page (the page reached when clicking on the search-site result) was 134 kilobytes, which is equivalent to approximately 19 seconds of download time on a 56 Kbps modem. Sites that were equal to or larger than 35 kilobytes (57%) were ranked significantly higher (tau = -0.39, P= .04) by the search sites. Eight (57%) and 11 (79%) of the sites had meta description and meta keywords tags, respectively. The presence of a meta description tag was significantly associated with a higher search rank (tau = -0.48, P= .002). Although 5 sites used cookies (small files sent to the browser along with a Web page for tracking a visit), only 3 of them used a persistent cookie that is stored on the user's hard disk and 4 used a session cookie that is automatically deleted from the browser's cache when the browseris closed. Six (43%) sites were just part of larger Web sites containing information other than smoking. The median link density of the 14 Web pages was 6 and the maximum was 735. A higher link density was significantly associated with a higher search rank (tau = -0.58, P= .02).
Discussion
The key finding of this study was that using simple search terms on popular search sites to look for information on smoking cessation for teenagers, less than half (14 of 30) of the sites found were of direct relevance. The remaining sites were study reports, news, and hyperlinks.
We did not include all information retrieved from Web searches, as has been done in studies on other topics [37], since users tend not to go beyond the first few pages of search results [9,10]. Instead, we evaluated only the top 30 search results to mimic typical Web search behavior.
Searching with the terms "teen quit smoking" on 7 popular search sites, Edwards et al [38] also reported that only 40% of the 140 potential hits were focused on cessation. In our study, 1 site of pornographic nature was found when using the search terms "teen smoking cessation" but no such sites were found when using the search terms "teen quit smoking" in contrast to a previous report [39] where 7 out of the top 20 sites were teen pornography sites.
Of public health concern was the finding that 3 sites were commercial sites and 2 of them were linked back to a single online drug store using a page-redirect spamming technique. With page redirection, an optimized page with unique and specific terms is submitted to search sites with the single purpose of ranking high on a specific topic. However, anyone clicking the link to this page is automatically sent to a real destination page, which often contains material unrelated to the initial search terms. For example, one site used "what-happens-to-your-body-when-you-quit-smoking.htm" as the name of its Web page. However, this page contains no information on smoking cessation. Instead, it is a page with a JavaScript that immediately redirects visitors to an online drug store.
Several important associations were found between Web-site characteristics and position ranking in the top 30 search results. These results can be used for optimizing site development in future smoking-cessation Web sites.
Essential-Characteristic Category
As an example of how these results can be used, of the 6 items in the essential-characteristic category, readability (lower grade level) was associated with higher position ranking. The lack of search box, navigational menu, privacy statement, or disclaimer, or the presence of broken links, was not uncommon, but their absence was not associated with lower position ranking.
Enhancement-Characteristic Category
In the enhancement-characteristic category, presence of contact e-mail address, medication-cessation information, alternative-approach information, and annotated external links were associated with higher position ranking. It is surprising to find that only 1 site displayed a HONcode insignia which, along with the associated membership, is an indication that a site complies with an 8-point code of conduct put forth by Health on the Net [18]. Although 73% of young people said that knowing who produced health information is very important to them, only 29% of those who looked up health information online checked the source the last time they conducted a search [5] and it is likely that fewer will check for the authenticity (for example, verify the membership status of a site at the HON Web site) of any indications of external recognition even if they are present [8].
Technical-Characteristic Category
In the technical-characteristic category, page size that was larger than 35 kilobytes, presence of a meta description tag, and a high link density were associated with higher ranking. The strong association between site description meta tag and ranking (tau = -0.48, P= .002) suggests that such information is relevant to the ranking algorithms of the search-engines used. Including a concise description tag is likely to be more effective in improving search-engine visibility than just a comprehensive keywords list. In fact, due to high rate of keyword repetition and spam, search sites such as Google and AltaVista do not give consideration to the keywords meta tag in their ranking [40,41]. As expected, link density is strongly associated with ranking (tau = -0.58, P= .02). Search engines generally use the number of incoming links (link density) in their ranking algorithm. However, Google's PageRank algorithm also takes into account the number of outgoing links on the page of each of the incoming links [42].Therefore, to achieve a high ranking a Web site should try to get listed on as many sites as possible and, in particular, on those sites that have as few external links as possible. Since search engines assign higher ranking to sites with incoming links that originate from pages containing fewer external links, and sites with annotated external links tend to have fewer links than those sites without annotated external links, this may explain the association between the presence of annotated external links and higher ranking (tau = -0.39, P= .04).
To improve search efficiency, users may want to supplement results from search sites with those from subject-based Web directories that are created and maintained by people, rather than by algorithms, such as Yahoo! Directory, which has a teen-smoking section [43]. Using the Yahoo! directory, we found 25 sites listed, of which only 4 were found using our search terms at the 4 popular search sites. In addition, users may want to learn and apply the specific syntax of their favorite search sites when searching for information. For example, quit-smoking Web sites of the commercial (.com) domain can be eliminated from the search results by entering "quit smoking -site:.com" in the search box in Google.

teens
teenagers
internet
smoking cessation
search engines
web page analysis