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MRP3: a molecular target for human glioblastoma multiforme immunotherapy.
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Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3).
We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS.
Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed MRP3 mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined MRP3-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of MRP3 RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002).
Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data suggest that the tumor-associated antigen MRP3 has potential use for prognosis and as a target for malignant glioma immunotherapy.
KeywordsMalignant Glioma MRP3 Protein MRP3 Gene MRP3 mRNA MRP3 Protein Expression
anaplastic astrocytoma
glioblastoma multiforme
Karnofsky performance score
fluorescence-activated cell sorter (analysis)
horseradish peroxidase
- MAb
monoclonal antibody
membrane-associated region
maltose-binding protein
multidrug resistance protein 3
reverse-transcription polymerase chain reaction
serial analysis of gene expression
Glioblastoma multiforme (GBM) is the most common and aggressive neuroectodermal neoplasm in adults. Although a recent study showed meaningful survival benefit associated with chemotherapy using a temozolomide-based chemoradiation approach [1], the median progression-free survival among patients treated with this regimen was only 7.9 months, and the overall survival was only 14.6 months [1]. Thus, an effective treatment for GBM patients is still a critical need.
Tumor-specific antigens that can be targeted by monoclonal antibodies (MAbs) conjugated with either radioisotopes or cytotoxins have great potential for cancer therapy [2, 3]. MAbs have been applied to the treatment of malignant gliomas through selective destruction of tumor cells and sparing of normal brain cells [4, 5]. Glioma-associated antigens targeted by immunotherapeutic approaches include cell adhesion molecules, matrix proteins, and growth factor receptors, such as tenascin [6], wild-type epidermal growth factor receptor [7], its glioma-associated variant, epidermal growth factor receptor variant III [5, 8], and GPNMB [9]. Notwithstanding the tumor-restricted presentation of these antigens, GBMs are a heterogeneous group of tumors, consisting of genotypically and phenotypically divergent populations of cells [10, 11]. As a result, antigenic expression profiles show a significant level of variation among and within individual GBMs [12]. Antigenic drift, observed in cultured cells, suggests the possibility of antigenic escape in primary brain tumors during treatment. Thus in any immunotherapeutic regimen, the antigenic heterogeneity seen in GBM necessitates the precise and timely selection of one or more target molecules for each patient. One approach to circumvent neoplastic cell heterogeneity is to expand the spectrum of GBM-specific targetable molecules and to customize therapy by using the best combination of targeted tumor antigens. Recent advances in genome technology have made it possible to analyze systematically the differences in gene expression patterns between normal and cancer cells, providing opportunities to discover novel antigens with tumor-specific distribution [13, 14].
Multidrug-resistance protein 3 (MRP3), also known as the ATP-binding cassette (ABC) superfamily C Member 3, or ABCC3, is an organic anion transporter that we have recently identified as a candidate GBM marker by the serial analysis of gene expression (SAGE) method [15]. The best-studied mechanisms of multidrug resistance in malignant cells involve the overexpression of ATP-driven anticancer drug efflux pumps of the ABC superfamily [16]. MRP3 is involved in ATP-dependent transport of hydrophobic compounds [17] and of bile acids under certain physiological conditions [18]. MRP3 has limited distribution in human normal tissues and is expressed in adrenal gland, kidney, placenta, and organs of the gastrointestinal tract, including intestine, pancreas, liver, and gallbladder [19, 20]. Recent work using MRP3-transfected cell lines has demonstrated the ability of MRP3 to transport certain classes of cytotoxic anticancer agents [21, 22, 23]. Accumulating evidence indicates that MRP3 gene expression is ectopically activated during carcinogenesis. MRP3, as mRNA or protein, has been detected in a variety of human cancer cell lines and tissues, including malignant gliomas [24, 25, 26, 27, 28, 29, 30], which implies possible involvement of MRP3 in the acquisition of a drug-resistant phenotype in these tumors.
Calatozzolo et al. showed that, in contrast to expression levels in nontumor brain samples, normal human astrocytes, and cultured endothelial cells, MRP3 is hyperexpressed in astrocytomas as the primary resistance to chemotherapy with drugs like cis-platinum (CDDP) and carmustine (BCNU) [31] and that MRP3 can modulate drug sensitivity to certain anticancer agents, such as cisplatin, vincristine, and etoposide, in human gliomas [25]. It has recently been shown that hepatic progenitor cells have high expression levels of functional MRP1 and MRP3, which may have a role in removing either exogenous or endogenous toxins and metabolites from progenitor cells [32]. Expression of ABC transporters was also found to be responsible for the highly enriched population phenotype in a wide variety of stem cells [33], and other initial studies also showed that they may be active in hematopoietic stem cells as functional regulators [34]. Although MRP3 was much less expressed in cancer stem cells before differentiation, after differentiation, the expression of MRP3 notably rose, which suggests that just after differentiation the cells acquired chemotherapeutic resistance via MRP3 [35].
In our preliminary survey of a limited number of glioma samples, MRP3 mRNA transcripts were highly expressed (80- to 100-fold induction) in comparison with normal brain tissues [15]. MRP3 could be an important target for immunotherapy, if the expression of MRP3 RNA by gliomas and minimum of expression in normal brain is reflective of actual MRP3 protein expression. This, along with its transmembrane localization, makes MRP3 an attractive target in immunotherapy for malignant gliomas. More pertinent, MRP3 is a tumor rejection antigen recognized by cytotoxic T lymphocytes in human lung adenocarcinomas [36]. However, little is known about MRP3 mRNA and protein expression in a large number of newly diagnosed GBM patient samples. We have therefore proposed to investigate the suitability of this MRP3 marker as a glioma immunotherapeutic target.
In this study, we conducted a genetic and immunohistochemical (IHC) evaluation of human gliomas to determine the incidence, distribution, and pattern of localization of MRP3 antigens in brain tumors. To assess the therapeutic potential of MRP3 as a GBM-associated antigen, we used real-time reverse transcription-polymerase chain reaction (RT-PCR) to determine MRP3 RNA transcript levels in 67 biopsy specimens from newly diagnosed GBM patients. In addition, a panel of 46 newly diagnosed GBM cases was assessed for MRP3 protein expression by IHC analysis, and survival analyses were performed based on these two parameters. We provide evidence that MRP3 mRNA expression is up-regulated in the majority of GBM cases and that glioma cells express MRP3 protein in vitro and in vivo. Increased MRP3 mRNA expression in GBM biopsy samples correlated with a higher risk of death. We demonstrate that MRP3 is a good potential molecular target for the immunotherapeutic treatment of malignant gliomas.
Cell lines
Human GBM-derived cell lines D54 MG, D247 MG, D392 MG, and D245 MG were established and maintained in our laboratory [10]; T98G and U251 MG were obtained from the ATCC. Glioma cells were grown in Zinc Option medium supplemented with 10% FCS (ZO-10% FCS). Cell lines with defined expression of MRP molecules were the kind gift of Dr. Piet Borst [21, 37, 38]. This defined cell line system consists of the nontransfected parent cell line 2008 (human ovarian carcinoma) transfected to express MRP1, MRP2, or MRP3. The HEK293 (human embryonic kidney) cell line was transfected to express MRP4. The MCDK (canine kidney) cell line was transfected to express MRP5. The monospecificity of MRP expression by this panel has been established [38]; all cell lines were grown in 10% FCS-DMEM (GIBCO/BRL 15050-022 [Invitrogen Corp., Carlsbad, CA]; with high glucose and phenol red) and split with 0.05% trypsin/EDTA.
For transient and stable expression, the cDNA of MRP3 was ligated into the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA). The resulting pcDNA3.1-MRP3 was introduced into HEK293 cells by using the Lipofectamine 2000 reagent (Invitrogen), and transiently transfected cells were harvested 48 h later. Stably transfected clones were selected in medium containing 200 μg/ml of Zeocin (Invitrogen).
Brain tumor samples
Patient Characteristics
Patient characteristic
All patients
Patients with MRP3 IHC Data
Patients with MRP3 mRNA Data
# of Patients
59 (63%)
30 (65%)
39 (42%)
35 (37%)
16 (35%)
28 (59%)
≤ 45 years
22 (23%)
10 (22%)
13 (19%)
>45 years
72 (77%)
36 (78%)
54 (81%)
51 (54%)
23 (50%)
38 (57%)
70 - 80
36 (38%)
21 (46%)
24 (36%)
50 - 60
5 (5%)
2 (4%)
3 (4%)
2 (2%)
2 (3%)
Extent of Resection:
75 (80%)
34 (74%)
55 (82%)
17 (18%)
10 (22%)
12 (18%)
2 (2%)
2 (4%)
Post-operative Treatment:
2 (2%)
2 (4%)
1 (1%)
2 (2%)
2 (4%)
1 (1%)
1 (2%)
4 (4%)
2 (4%)
2 (3%)
7 (7%)
5 (11%)
7 (10%)
4 (4%)
2 (4%)
2 (3%)
Mab I131-81C6
8 (9%)
5 (11%)
7 (10%)
3 (3%)
2 (4%)
2 (3%)
1 (1%)
1 (1%)
1 (1%)
1 (1%)
1 (1%)
1 (2%)
19 (20%)
12 (26%)
15 (22%)
37 (39%)
11 (24%)
26 (39%)
4 (4%)
1 (2%)
3 (4%)
Survival Status:
7 (7%)
3 (7%)
5 (7%)
87 (93%)
43 (93%)
62 (93%)
Available Data:
RNA only
48 (51%)
48 (72%)
IHC only
27 (29%)
27 (59%)
RNA and IHC
19 (20%)
19 (41%)
19 (28%)
Quantitative RT-PCR assay
Total cellular RNA for use in the quantitative RT-PCR assay was isolated from confluent cultured cells and tumor tissues by using an RNeasy Mini Kit (Qiagen, Valencia, CA) and then treated with RNase-free DNase I (Ambion, Austin, TX). Total RNA of normal adult whole brain was either purchased from Clontech (Palo Alto, CA) or obtained from the Brain Tumor BioRepository, Preston Robert Tisch Brain Tumor Center, Duke University Medical Center. These total RNA samples (0.2 μg) were converted to random-primed cDNA with SuperScript II RNaseH- Reverse Transcriptase (Life Technologies, Rockville, MD) in a reaction volume of 20 μl. After reverse transcription, 280 μl of water was added to the reaction mixture, and 2 μl of diluted cDNA sample was used as a template in PCR experiments.
The synthesized first-strand cDNA samples were subjected to real-time PCR with SYBR Green dye. Incorporation of SYBR Green dye into the PCR products was monitored with an ABI PRISM 7900HT Sequence Detector System (Applied Biosystems, Foster City, CA). For PCR reactions, 2 μl of cDNA, 5 μl of 2× SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK), and 200 nM of each primer were used in a total volume of 10 μl. Cycling parameters were 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 64°C for 30 s. For each sample, β-actin transcript was amplified simultaneously as an internal control. To avoid amplification from contaminating genomic DNA, primer pairs were designed to amplify sequences spanning introns. The oligonucleotide primer sequences were as follows.
MRP3 (sense) 5'-TGCCATCGACCTGGAGACTGACAAC-3'
MRP3 (antisense) 5'-GACATATTTGGTGTCATTTCCTTCCTGATG-3'
β-actin (sense) 5'-CCAACCGCGAGAAGATGACCCAGATCATGT-3'
β-actin (antisense) 5'-GGTGAGGATCTTCATGAGGTAGTCAGTCAGG-3'
In each tumor, relative MRP3 mRNA expression levels were measured in terms of fold induction ratio over normal whole-brain sample, which was determined by dividing the MRP3/β-actin ratio of the tumor sample by that of control normal whole brain. All measurements were performed in triplicate wells and repeated twice. Positive cases were defined as those with MRP3 RNA levels 3-fold higher than normal brain.
MRP3 fusion protein
sense: 5'-TCGGATCCGGCACCATGGCTGCCATTGGAGCCAATGTG-3'
antisense: 5'-AGTCTAGATTACCCCGGGTTCAGGCTGCTCCT-3'
After digestion with BamHI and XbaI, the MAR3 DNA fragment was cloned into the corresponding restriction site of pMAL-c2X. The cDNA fragment for the MAR3 domain was also amplified by RT-PCR by using the same PCR primers and total RNA isolated from human glioma cell line T98G as a template. DNA sequencing revealed that nucleotide sequence of the MAR3 segment of MRP3 expressed in T98G was the same as that in pGEM7Zf-MRP3 and in previous reports [21, 41] (data not shown). The resulting fusion protein MAR3-MBP was produced in E. coli TG1 and purified from cytosol medium with amylose resin affinity chromatography according to the manufacturer's instructions.
Rabbits were given a primary s.c. immunization with 250 μg of recombinant protein MAR3-MBP emulsified 1:1 in Freund's complete adjuvant and were subsequently boosted with the same dose of antigen emulsified 1:1 in Freund's incomplete adjuvant at 4-week intervals. Serum titers were determined by ELISA versus MAR3-MBP or MBP alone as target antigens and by live-cell-ELISA using MRP3-expressing D247 MG cells as targets [8]. Sera obtained 12 days following the sixth immunization were passed over a staphylococcal protein A column as previously described [42], and the rabbit immunoglobulin concentration was determined by capture ELISA [42].
Balb/c mice (NCI Animal Production Program, Frederick, MD) were similarly immunized with protein MAR3-MBP on days 171 and 191, following a four-dose immunization regimen with pcDNA3.1-MAR3 over days 1--87; the DNA-alone immunization protocol failed to produce titers sufficient to warrant fusion. Following MAR3-MBP protein boosts, 50% endpoint titers to target were in excess of 1/10,000, and spleens of two mice were fused. MAbs were initially identified for positivity with MAR3-MBP and absence of reactivity with MBP by ELISA; subsequently, they were further screened on cell lines that were determined, by mRNA analysis and reactivity with the polyvalent rabbit anti-MAR3-MBP serum, to be MRP3 positive (Western blot and indirect immunofluorescence analysis). Anti-MAR3 MAb 16A11, of the IgG2a isotype, was chosen because of its superior reactivity in indirect FACS analysis with MRP3-expressing D54 MG cells.
Western blotting
Cells were lysed in NP-40 lysis buffer (50 mM Tris-Cl, pH 8.0/150 mM NaCl/1% NP-40/1 mM phenylmethylsulfonyl fluoride/0.045 mg/ml aprotinin). Aliquots of 10 μg of total protein were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were incubated with 3% non-fat milk in PBS-0.1% Tween 20 to block nonspecific binding and were then probed overnight at 4°C with rabbit anti-MAR3 antiserum 1708 in PBS-0.1% Tween 20 containing 1% milk. The protein bands were detected by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody coupled with SuperSignal West Pico Chemiluminescence Kit (Pierce, Rockford, IL).
Indirect FACS analysis
Flow cytometric analysis for MRP3 expression was performed as described previously [43]. Test reagents and wash buffers were kept on ice to ensure the detection of cell surface molecules without allowing internalization to occur. Target cells were detached from culture flasks by incubation with 0.02% EDTA/PBS. Then, 1 × 106 cells/reaction mixture were maintained in 0.5% paraformaldehyde/PBS for 10 min at 4°C, washed, resuspended in 150 μl of PBS containing 10% FBS, and blocked for 20 min at 4°C. After two washes, the samples were reacted with rabbit anti-MAR3 1708 purified IgG, normal rabbit IgG, MAb 16A11, or murine IgG2a (all at 10 μg/ml in PBS) and rotated for 1 h at 4°C. After two additional washes, cells were incubated with species-appropriate FITC-labeled secondary antibody for 30 min at 4°C and then analyzed on a Becton Dickinson FACSort flow cytometer and cell sorter system (Becton Dickinson, San Jose, CA).
Quantitative FACS analysis
We have previously published our techniques for QFACS analysis in the EGFRvIII MAb-antigen system [43]; briefly, FITC-MAb 16A11 is reacted with a cocktail of beads (Bangs Laboratories, Inc., Fishers IN) which bind no (B1) or graded amounts (20,000-200,000 [B2-B5]) of murine IgG molecules. Regression analysis of bead-binding capacity versus FL1 height is performed, generating a straight line formula, from which we calculate the number of molecules of FITC-MAb 16A11 bound to the target cells of identical aliquots, assuming 1:1 stoichiometry.
Anti-MRP antibodies
All anti-MRP reagents were obtained from Kamiya Biomedical Company (Seattle, WA). Anti-MRP1 (MRPm6, IgG1), anti-MRP-2 (M2II-12, IgG2a), and anti-MRP3 (M3II-9; IgG1) are all murine monoclonal reagents directed against the cytoplasmic domain epitopes of their respective targets. Anti-MRP4 (M4I-10) and anti-MRP5 (M5I-1), from the same source, are rat IgG2a MAbs, also directed against a cytoplasmic domain epitope.
Analysis of cells grown on chambered slides
Target cells were plated at densities determined in previous assays to yield a 70-80% confluent monolayer in either 24 or 48 h. Our IHC procedure using Lab-Tek Chamber Slides (Electron Microscopy Sciences, Hatfield, PA) has been published previously [43]; however Triton X treatment was omitted, as detection of nuclear staining was not an objective. Due to the cytoplasmic location of the epitopes detected by commercial reagents, all assays were performed in the presence of saponin, to allow penetration of reagents to the cytoplasm. Following fixation, blocking, and permeabilization, primary reagents (all at 12.5 μg/ml) were applied, and the assay continued as described, with the exception that the secondary reagent used was biotinylated horse anti-mouse IgG (Zymed, San Francisco, CA) or biotinylated goat anti-rat IgG (Caltag) as appropriate. Slides were counterstained with hematoxylin and read by two independent observers.
Immunohistochemical staining of frozen sections derived from human glioma tumors was performed as described previously [43] using rabbit polyvalent anti-MAR3 antiserum 1708 purified IgG. Briefly, frozen tissues were sectioned (5-7 mm), mounted on positively charged slides, air-dried for 30 min, and fixed in cold acetone for 30 s. Nonspecific protein binding sites were blocked at room temperature with PBS containing 10% normal goat serum for 30 min, followed by incubation with primary antibodies at 10 μg/ml for 2 h. Positive reactions were detected by using biotinylated goat anti-rabbit IgG and HRP-conjugated streptavidin (Zymed). Bound peroxidase was developed with diaminobenzidine for 5 min. As a negative primary reagent control, irrelevant normal rabbit immunoglobulin (10 μg/ml) was used.
Slides were counterstained with hematoxylin, dehydrated, mounted, coded, and read by two investigators; staining patterns were scored as focal, multifocal, or diffuse, both in perivascular and parenchymal distribution, with intensity of stain described as weak, moderate, or strong.
Statistical analyses
Cox's proportional hazards model was used to examine the effect of MRP3 mRNA expression (≤10-fold vs >10-fold) and IHC (zero vs positive) on survival, which was computed from the date of pathology sample acquisition to date of death or last contact. Patient characteristics such as gender, age, KPS, extent of resection, histology, post-operative treatment, and survival status are shown in Table 1. Analyses were conducted with and without adjustment for known prognostic factors such as age (≤ 45 years vs >45 years) [9], extent of resection (GTR vs STR/biopsy), and KPS (90-100 vs <90). Backwards elimination was used to identify a parsimonious multivariable model (SAS statistical analysis package, Cary, NC).
Quantification of MRP3 mRNA in glioblastoma-derived cell lines and GBM samples
Incidence of MRP3 mRNA expression in GBM patient samples
From the whole series of 94 newly diagnosed GBM patients, we measured MRP3 mRNA levels in 67 GBM biopsy samples. MRP3 mRNA expression was measured by quantitative RT-PCR in tumor samples and compared to that of normal whole brain (Fig. 2). Positive cases were defined as those with MRP3 RNA levels 3-fold higher than the level in normal brain. We found that 59 (88%) of the 67 GBM samples were positive, the MRP3 mRNA induction ranging from 3.5- to 370-fold over that in normal brain. Significantly, 43 (64%) of the 67 cases had expression of MRP3 mRNA greater than 10-fold over that seen in normal brain RNA extracts (data not shown). The expression levels of MRP3 transcripts were correlated positively with the grade level of gliomas (data not shown). Only minimal expression for the MRP3 gene was detected as a definable baseline in normal brain samples assayed simultaneously.
Detection of MRP3 protein in glioblastoma cells
Quantitative FACS analysis of MRP3 expression by cell lines, biopsies, and established xenografts with MAb 16A11
Quantitative FACS analysis of MRP3 expression by biopsies, cell lines, and established xenografts with MAb 16A11
Positive cases/Total cases
Estimated number of MRP3 molecules per cell
Cell Lines
GBM adult
0.1-4.9 × 105 (med. = 1.9 × 105)
MED pediatric
0.7--2 × 105 (med. = 1 × 105)
AA adult
1.7--2.7 × 104
GBM adult
0.17--6.5 × 105 (med. = 6.5 × 104)
GBM adult
0.2--2.1 × 105
MED pediatric
1.6--2.5 × 104
We used FITC-MAb 16A11 with QFACS analysis to investigate MRP3 density on the cell surface of the cultured glioma cell lines shown to express MRP3 mRNA--D54 MG, D247 MG, T98G, U251, and D392 MG--and on human GBM biopsy samples. D54 MG cells were relatively homogeneous in expression of MRP3 on the cell surface (Fig. 3C), with a median density of 4.4 × 104 molecules per cell (6 repeat assays). D247 MG similarly expressed a median density of 2.3 × 104 molecules per cell, T98G expressed 0.38--4.9 × 105 molecules of MRP3 per cell (8 repeat assays; data not shown), U251 MG cells exhibited 0.4--2.1 × 105 MRP3 molecules per cell (3 repeat assays; data not shown), and D392 MG was uniformly negative for cell surface expression of MRP3 (4 repeat assays; data not shown).
Biopsies and xenografts of GBM tended to express lower cell surface MRP3 densities (median values in the 104 range) than did cell lines (median = 1.9 × 105). Of six adult anaplastic astrocytoma (AA) biopsies analyzed (single assay in triplicate on freshly isolated cells), three were positive for cell surface MRP3, with a range in expression of 1.7--2.7 × 104 MRP3 molecules per cell (Table 2). Significantly, 22/27 adult GBM biopsies (82%) were positive for cell surface MRP3, with a range in expression of 0.17--6.5 × 105 (median = 6.5 × 104) MRP3 molecules per cell (Table 2). Representative data from TB 2155 MG is shown in Fig. 3D (density/cell: 2.7 × 104); again, the density distribution by these biopsy-derived cells is relatively homogeneous. This tendency was also observed in the medulloblastoma cell lines and xenografts examined. Of the six adult AA biopsy samples that were available for analysis, only 50% were positive, as opposed to 82% for GBM biopsies. Unlike the EGFRvIII variant, MRP3 appears to be maintained over time in cell culture. The slightly lower frequency of expression in established GBM xenografts (57%) may be preliminary due to the small sample size (7 cases).
Immunohistochemical analysis of defined cell lines variously transfected to express MRP1-5
Immunohistochemical analysis of defined MRP-expressing cell lines*
Negative Control
Anti-MRP3 (M3II-9)
Anti-MRP3 (16A11)
Anti-MRP3 (1708)
Immunohistochemical analysis
Summary of immunohistochemical evaluation of frozen GBM tissue sections with polyvalent anti-MRP3 antiserum 1708
Reactivity patterns
Parenchymal and perivascular
Total reactive
Statistical analyses
Multivariable survival models were also run for survival analyses. Backwards elimination was used to generate these models while retaining either the IHC or the mRNA predictor in the model. In these multivariable analyses, IHC positivity is not predictive of survival (p = 0.723). However, after adjustment for extent of resection, high levels of RNA remain a significant predictor of survival (p = 0.001; HR = 2.54 with 95% confidence interval 1.44, 4.49).
In an ideal situation, target antigens for passive immunotherapy should be (1) tumor specific, that is, expressed in tumor and not normal tissue and (2) accessible (cell surface or matrix) at a density sufficient for targeting and significant cell kill. The criterion of absolute tumor specificity is rarely met, and current approaches often must target tumor-associated antigens (ratio of neoplastic tissue to normal tissue expression acceptable for lack of bystander effect).
In the present study, we have demonstrated that the MRP3 gene is expressed both at the mRNA level and the protein level in high-grade gliomas, but minimally in normal brain tissue. By quantitative RT-PCR analysis of 67 newly diagnosed GBM patient samples for MRP3 mRNA expression, 88% of the samples were positive, and 64% showed levels 10 times greater than the level of MRP3 mRNA expression in normal brain. Immunohistochemical analysis of 46 cases revealed that 91% of the GBM samples expressed MRP3 protein detectable by immunohistochemistry. The predominant staining pattern in all GBM was either parenchymal distribution only (22/46, 48%) or mixed perivascular and parenchymal in orientation, either focal or diffuse in distribution (18/46, 39%), with few cases showing only prominent perivascular distribution (2/46, 4%). As for the cellular origin of MRP3 expression in the bulk tumors, five of six GBM-derived cell lines examined in this study were positive for MRP3 mRNA transcripts (Fig. 2), this result supporting the view that MRP3 is produced mainly by transformed cells in glioma tissues.
Our results and those of other researchers indicate that MRP3 is highly expressed at gene and protein levels in human glioma cell lines and in clinical glioma specimens of grades III and IV [25]; consistent with other studies, we observed no apparent expression of MRP3 in normal brain tissue [45]. In addition, of the MRP family genes, MRP1 was also found to have increased mRNA expression in some human glioma cell lines and clinical samples [24, 25]. However, MRP1 is known to be expressed in normal brain tissue [27]. MRP4 and MRP5 have been reported to exhibit expression in two glioblastoma cell lines, whereas MRP2 was not expressed in malignant glioma cells [24, 25]. Bronger et al. have also detected the expression of other MRP family members, such as MRP4 and MRP5, in the blood-brain barrier and in the glioma cells at protein level [46]. Localization of MRP1, MRP4, and MRP5 protein in rapidly frozen perilesional samples of several regions of adult human brain was reported by Nies et al. [47]. Among normal vital organs outside the CNS, MRP3 protein expression has been observed in adrenal gland, kidney, and tissues associated with the circulation of bile acids [20].
However, the expression of MRP3 in normal tissues outside the CNS will not compromise the compartmental delivery of MRP3-related immunological agents within the CNS, because the optimal route for the administration of MAb-based therapeutic agents for tumors localized within the CNS is through surgically created resection cavities or saturation of an entire hemisphere by intracranial microdiffusion, called convection-enhanced delivery, to the brain tumor [48], which allows direct parenchymal infusion of therapeutics, bypassing the blood-brain barrier. Only trace amounts of therapeutics distribute systemically, and the possibility of life-threatening side effects, such as lung edema, would be minimal. However, these focal deliveries have limitations: Passive microdiffusion happens to only a very poor extent, given the gliosis reaction in the wall of the surgical cavity, and convection-enhanced delivery must take into account modern calculations for isodose delivery distributions to cover the relevant areas [49].
Localization to the tumor cell surface membrane or extracellular matrix is another prerequisite for target antigens in MAb-mediated therapy [50]. Amino acid sequence analysis has shown that in a topological model, MRP3 comprises seven domains, three of which contain multiple extracellularly expressed regions, while the remaining four are intracellularly located [40]. Although MRP3 protein is predicted to integrate in the cell surface membrane, the precise membrane topology of MRP3 is complex and remains unclear. Depending upon the software used, 16-18 transmembrane domains are predicted for MRP3, which complicates finding the precise location of extracellular epitopes of MRP3 [40]. To address this, our immunization was performed with fusion protein MAR3-MBP (MAR3; amino acids 984-1224 of MRP3), which may contain extracellular epitopes of MRP3, and serum titer was monitored by the reactivity with MRP3-expressing GBM cells under conditions detecting cell surface reactivity only. The resulting rabbit antiserum 1708 and murine MAb 16A11, derived from immunization with MAR3-MBP, recognized cell-surface-expressed MRP3 as defined by immunofluorescence analysis of MRP3-positive GBM cells under non-fixed conditions. These results from flow cytometry, along with IHC findings showing membranous staining of tumor cells, indicate the expression of MRP3 protein on the surface membrane of glioblastoma cells in vitro and in vivo. Recently, we have isolated scFvs M25 and M58, which specifically react with the extracellular N-terminus of MRP3, for IHC evaluation of human gliomas to determine the localization of MRP3 antigen. These Fv-based recombinant antibodies, possessing superior tumor penetration capabilities and selectively targeting tumor cells that express MRP3, may potentially be used in immunotherapy and diagnosis for brain tumors and other cancers [51].
Other factors that define the targetability of antigen molecules involve antigen density [52] and stability (lack of antigen shedding and internalization following binding to ligand or antibody) [50]. Results from immunotherapeutic experiments using xenograft models have revealed that the extent of tumor reduction elicited by a single MAb is generally proportional to the antigen density at the cell surface [52]. Although the minimum antigen density required for successful MAb-mediated therapy varies, depending on the mechanisms of action of cytotoxic conjugates, the general consensus is that ≥1 × 104 protein molecules per cell would be required for biologically effective targeting [43]. Preliminary data obtained by quantitative FACS analysis with MAb 16A11 directed against MAR3-MBP indicates that human malignant glioma cell lines T98G, D247 MG, and D54 MG express 0.3-4.9 × 105 MRP3 molecules per cell. More significantly, biopsy-derived GBM cells from 22/27 patients expressed MRP3 at a density ranging from 1.7 × 104 to 6.5 × 105 molecules per cell, well within the density range required for targeting.
The molecular mechanisms underlying overexpression of the MRP3 gene in gliomas remain to be determined. Activation of the MRP family genes can be achieved in several ways. For MRP1, an increase in MRP1 mRNA in cultured cell lines that have been selected for in vitro drug resistance is frequently associated with amplification of its cognate gene and at least some level of rearrangement [53], while elevated MRP1 mRNA found in human tumor samples occurs without [54] or only rarely with gene amplification [55]. Thus, further investigation could determine whether MRP3 mRNA overexpression in human gliomas also is associated with an increase of gene copy number. Up-regulation of MRP3 mRNA can occur by mechanisms other than gene amplification. Sequence analysis of the 5'-flanking region of the MRP3 gene has revealed that the MRP3 promoter lacks the typical TATA box and contains multiple potential binding sites for a number of transcription factors, including AP1, AP2, N-MYC, Sp1, and PEA-3 [41, 56]. Alternatively, methylation of the promoter region, the stability of mRNA, or translational efficiency may be altered, which can contribute to the hyperexpression of MRP3.
The biological significance of aberrant expression of MRP3 in glioma tissues is not yet known. Our results and reports from other researchers [24, 25] have demonstrated that MRP3 mRNA levels increase with tumor grade, indicating that MRP3 is likely to be a potential progression marker of glial tumors. In human colorectal carcinoma cell lines, the enhanced expression of MRP3 mRNA is associated with the inactivation of the p53 gene [57]. MRP3 gene expression may be regulated at the genetic level, depending on the status of oncogenes and/or tumor suppressor genes involved in the tumorigenesis of gliomas. On the other hand, one of the outstanding features of MRP3 is its inducibility by external stimuli, such as chemical carcinogens and anticancer drugs. This inducible nature of MRP3 has been observed in primate and rodent tissues and in a variety of cultured cells [58, 59], and drug-induced MRP3 may provide an important mechanism for cytoprotection from the toxicity of xenobiotics. However, glioma cases examined in this study exhibited MRP3 overexpression prior to the exposure to antineoplastic reagents. MRP3 can also be up-regulated in response to a change in the intracellular redox state [57] and, as postulated for MRP1 [60], may improve cellular defense status against oxidative metabolites that accumulate during tumor growth.
The categorical variables of age, mRNA transcript level, and IHC staining intensity were used to construct Kaplan-Meier curves, and our univariate analysis demonstrated efficacy in predicting survival in the newly diagnosed GBM population. The univariate analyses show that patients with high MRP3 mRNA expression level had a higher risk (2.71 times higher) of death than the patients with low/moderate MRP3 mRNA expression level. The results of survival analysis suggest that the relative MRP3 mRNA levels represent a potential prognostic predictor of poor GBM patient survival. There are only a few molecular markers that are prognostic for survival in malignant gliomas. Elevated expression of MRP3 in GBM patient specimens, strong GST-π protein expression in human gliomas [61], a functional polymorphism in EGF [62], and high GPNMB expression in GBM patients [9] are associated with clinically more aggressive gliomas and are useful and powerful prognostic markers of poor patient survival.
In conclusion, human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data indicate that the tumor-associated antigen MRP3 has potential use as a prognostic predictor for malignant gliomas. We further demonstrate that immunotherapeutic strategies designed to target MRP3 may be successful for treating malignant glioma patients.
We thank Dr. Marcel de Haas (The Netherlands Cancer Institute, Amsterdam, The Netherlands) for providing the pGEM7Zf-MRP3 vector. We also thank T. Shelley Davis for Western blot analysis, R. Ian Cumming for quantitative FACS assays, David Lister for RNA preparation, Dr. Joanne Ayriss, Shawn Connelly, and Ling Wang for IHC analyses, and Jeannette Day for statistical analyses. This work was supported by the following NIH Grants to Dr. Bigner: NINDS Grant 5P50 NS20023, NCI SPORE Grants 5P50 CA108786, NCI Merit Award R37 CA011898, and Grant MO1 RR 30, General Clinical Research Centers Program, National Center for Research Resources, as well as an FCG grant to Drs. Kuan and Wikstrand.
Supplementary material
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Friday, March 25You're Looking Happily Deranged.
Any longtime fan of the CDP will know that I have spent countless hours ranking, listing, scrutinizing, championing and singing the praises of 90's television. My love of recent nostalgia knows no boundaries or shame, and thanks to Social Networking, millions of others have also given a digital thumbs-up to similar shows of the era. Advertisers finally saw dollar signs at the prospect of re-airing these shows, and thanks to their greed and lack of original ideas, we now have a whole slew of old (but awesome) television shows to look forward to in 2011.
Here are a few of my favorites.
WHAT? – 'The 90's Are All That' Programming Block
WHERE? – TeenNick
WHEN? – Fall 2011
When I was a teen, Nick at Nite played I Love Lucy and The Honeymooners. In more recent years, they've filled the generation gap with shows like Full House and Family Matters. With 'The 90's Are All That,' we'll see such SNICK favorites as The Adventures of Pete & Pete, The Amanda Show, All That, Kenan & Kel, Rugrats and Clarissa Explains it All. I'm assuming (and hoping) that if this block becomes a hit, they will expand it with more classics from the era (Are You Afraid of the Dark? and The Secret World of Alex Mack spring to mind).
WHY THIS IS AWESOME – SNICK was good times for a teen. The big orange couch. The Saturday night timeslot. The feeling that you were in on something special; something that was made just for you. The '166 hours' closing always bummed me out, as it reminded me that Sunday was tomorrow and I'd have to prepare for another week of school, but for those few hours, SNICK made everything better. It reminds me of a fun time in my teen years, and if they add some retro bumpers and advertisements (TV Land did this in their youth), it could really be something fun and popular.
WHY THIS COULD BE AWFUL – If they 'enhance' these episodes with facts, pop-ups and tidbits, I'll be a very upset man. If you're bringing back a nostalgia block, keep it nostalgic. Also, most of the shows they're bringing back are for a slightly younger-than-teen audience.
WHAT? – 120 Minutes
WHEN? – Sometime in 2011
In my opinion, 120 Minutes was the best video-oriented show MTV ever had (next to Alternative Nation, perhaps). In recent years, MTV2 has aired Subterranean to fill the gap, but it's always been missing something (VH1 Classic airs a similar show, also titled 120 Minutes, but it's still lacking in many ways). With fantastic longtime host Matt Pinfield returning to the helm, it's good to see that MTV2 is going back to their Alternative roots, even for just two hours every few weeks.
WHY THIS IS AWESOME – The first few years of MTV2 (M2 at the time) were absolutely fantastic. It was as delivered: 24 hour-a-day, left-of-dial videos by up-and-coming bands with extremely limited VJ banter and absolutely no scripted (or unscripted) programming to speak of. Eventually, MTV2 became nothing more than an MTV clone, but with the return of 120 Minutes, we could see an advent of more retro, return-to-basics programming. Okay, maybe not. But this is still cool.
WHY THIS COULD BE AWFUL – While I don't expect 120 Minutes to fully operate as a nostalgia act, I do hope they air videos from the heyday of the show. Spending all of their time on new bands and videos could instantly alienate the very audience they're trying to attract (which I presume is former MTV viewers).
WHAT? – Beavis & Butthead NEW EPISODES
WHEN? – Summer of 2011
When King of the Hill ended, Mike Judge apparently had nothing to do. Fortunately, he decided to revive his two most popular characters, leading to a Summer re-launch of a decade-defining series. From what I can gather, the animation, voices and format will be identical to the original, with B&B riffing on newer music videos as well as whatever else they find on their television (viral videos, for example).
WHY THIS IS AWESOME – Mike Judge. He cannot fail (even when he does). He understands satire better than anyone, and if he thinks he can create something that will compare to what he did with Beavis & Butthead in the past, I will not doubt him for the time being. Furthermore, in an era that has moved beyond a lot of the cynicism and toilet humor that made them popular, it somehow makes Beavis & Butthead more relevant (and needed) than ever.
WHY THIS COULD BE AWFUL – As is the case with any revival, you run the risk of not living up to unrealistic expectations, or just the expectations of your former material. Also, the idea of making fun of new videos seems interesting, but bringing other current material into the mix might be strange and lackluster for fans expecting the same product they got in 1994.
WHAT? – Daria
WHERE? – Logo
WHEN? – Right Now!
Daria, the Beavis & Butthead spinoff, was a more cerebral, female-friendly take on the cliques and cultures of 90's suburban teenagers. Daria became an iconic anti-hero of sorts, and it's easy to argue that this was a better overall show than B&B. After airing reruns on The N for a few years, it has found a new home at the MTV-owned Logo Network.
WHY THIS IS AWESOME – I feel that Daria never got its due; an extremely underrated and intelligent show about the sometimes-nihilist world of 90's teenagers. In a 2010 review of the DVD collection in the website Jezebel, Margaret Hartmann said that at as a teenager, "Daria and her best friend Jane Lane provided me with the sort of social guidance that allowed me to stay true to myself" and led to her keeping a childhood friend instead of dropping her to avoid "social suicide." "I'd picked up Daria's attitude that it's easier to survive high school with one fellow-loser who shares your misanthropic views than to spend four years trying to earn the admiration of girls whose main interests include proper eyeliner application." She cites Daria as "the most authentic TV nerd... she didn't look for her fellow students to accept her. She just wanted to be left alone." DVD Talk's review referred to the show as "an indictment of everything MTV now embraces."
WHY THIS IS AWFUL – A lot of these episodes have been shortened for syndication, and nearly all of the original music has been removed and replaced, essentially suffering the same fate that Beavis & Butthead, The Wonder Years and many others have in the DVD age. Still good, though.
WHAT? – The Wonder Years
WHERE? – Hub
WHEN? – Right Now!
One of my favorite shows of all-time, The Wonder Years probably shaped my storytelling and narrative voice more than any book or movie. This show, easily the best series never properly released on DVD (more on that later), holds up wonderfully, capturing a youthful, nostalgic adolescence better than just about anything before or since. Although never released on DVD, full episodes have been brought back to television, thanks to the Hub Network.
WHY THIS IS AWESOME – It's The Wonder Years. Either you already love it, or you haven't watched it yet.
WHY THIS IS AWFUL – Just like Daria, the licensing for certain songs kept the show from receiving a proper DVD treatment, and the later episodes with a more grown-up Kevin Arnold are slightly lacking in tone. That being said, it's not nearly enough to avoid it. I recommend this with every fiber of my mid-life crisis-ravaged being.
So, there you have it. A handful of shows from the 90's that are either on their way back in syndication, back in new episodes, or already back on obscure cable networks. Sound off in the comments section, enjoy your day, and let me know what show you'd like to see return to the airwaves.
Glad to have you back dude! We need to get together for beverages and marches! Bring TJ, Emily, the wife and the rest of the Hitler Youth (heh...)<< Home
Not the 90's, but WKRP suffers on DVD for a lack of license for the songs, which loses a lot of the effect. Then there's the whole saga of URGH! A Music War.
The best part of Beavis & Butthead were the videos (I remember that's how I found out about Helium) so the DVDs were lame because of that.
I always wanted to have a 3-way with Daria and Jane. Can you have ironic, laconic orgasms?
Not the 90's, but WKRP suffers on DVD for a lack of license for the songs, which loses a lot of the effect. Then there's the whole saga of URGH! A Music War.
The best part of Beavis & Butthead were the videos (I remember that's how I found out about Helium) so the DVDs were lame because of that.
I always wanted to have a 3-way with Daria and Jane. Can you have ironic, laconic orgasms?

subset: commoncrawl


text:
== Tram Station ==
As Isaac steps off the ship, --THE VALOR-- database file will play. Open the
--ITEM CHEST-- to the left, stomp the --ITEM BOX-- in the sitting area then use
the !!UPGRADE BENCH!! directly ahead if necessary. Move down the hall to the
[ ] --"The Valor" database file--[ ] Item Chest[ ] Item Box
== Zero-G Ore Storage Hallway ==
Further down the hall, an --ITEM CHEST-- will be to the left of a !!STORE!! and
an --ITEM BOX-- will be in front of the next door at the end of the hall.
into the next room. Open the --ITEM CONTAINER-- on the wall straight ahead
then open the next door.
[ ] Item Chest[ ] Item Box[ ] Item Container
== Ore Storage ==
The --RADIATION HAZARD-- database file will be received shortly after entering
the room and a new objective will be added.
[ ] --"Radiation Hazard" database file--
Objective Added:
[ ] Clear Radioactive Material (0/6)
One of the Valor's weapon modules ripped open in the crash scattering
highly radioactive munitions through the Ore Storage chamber. The
radiation leak has ceased the Valor's airlock to seal itself. I need to
clear the chamber of the munitions before I can get onboard the ship.
This area does not have any gravity, so Isaac can move just about anywhere in
here. Before you can dispose of the green radioactive circular objects,
must first open the door in the middle of the room. Walk toward the middle
door on the floor and stomp each of the six purple fuses around the door -
there are three on each side. There are two --ITEM BOXES-- floating around
this room (near the door) so look around the room for them before stomping the
[ ] Item Box[ ] Item Box
The room will enter a vacuum as all the air is drained out when the middle door
opens. A Stinger and a Lurker will attack directly afterwards.
Be sure to
take note of the two oxygen rechargers around the middle door. Eventually two
Dark Stingers and two more Lurkers will attack while Isaac moves around in this
Look around the room and grapple each of the six green radiation balls with
kinesis then bring them down to the open door on the floor in the middle of the
room and then blast them out into space.
You will get a "Radioactive material
removed" confirmation when each of them is tossed out into space. Three of the
radioactive balls are up on a wall with circular metal pieces on it to the
left. Some of the radioactive balls are hidden behind the circular metal
Objective Complete:
[X] Clear Radioactive Material (6/6)
Once all of the green radiation balls have been thrown out into space the door
on the side of the UMG Valor will open. Look up on the ship and find the
circular ring of orange lights then jump to it and open the door on the walkway
in between it.
== Starboard Corridors ==
Objective Complete:
[X] Enter the Military Ship
Open the next door once the door behind Isaac is sealed off and the air is
Gee god! What the hell was that up ahead? It just broke into a spasm then ran
to the left!
Anyway, open the --ITEM CHEST-- off to the right. The --FIND THE
SINGULARITY CORE-- database file will play eventually.
[ ] Item Chest[ ] --"Find the Singularity Core" database file--
Objective Added:
[ ] Find the Singularity Core
Made it inside. Now I just have to find a way to the engine room and
salvage the Singularity Core.
Avoid the flame up ahead or Isaac will get burned. Move by the glowing reverse
gravity panel and save your game at the !!SAVE STATION!! on the wall ahead.
Good thing they save their games on the USM Valor as well, eh? Enter the Cargo
== Cargo Bay ==
Oh, great. There's that crazy thing up ahead once again!
It will run away
soon. Open the --ITEM CHEST-- off to the right. Walk over to the escape pod
and use kinesis to grapple the --RANDOM AMMO-- inside and pull it to Isaac then
move further ahead toward the door that is blocked by debris and open the
nearby --ITEM CHEST--.
[ ] Random Ammo[ ] Item Chest[ ] Item Chest
The --INFECTED SOLDIERS-- database file will be received when Isaac moves
further down the right side of this room. Move over to the right side of the
area with the cargo and find the first crate with the glowing kinesis symbol on
it. All the crates in this room are on tracks, so they can only be moved in
two directions. Grapple the crate with kinesis then move it further ahead past
the bigger crates. Try not to move it all the way back to where it pushes the
other crate with the kinesis symbol. Step into the section off to the left
then turn and grapple the crate you were just moving from the side then pull it
to the right - walk sideways to the right while it is grappled. Grapple the
next crate across from the one you just moved and move it to the right as well
by walking sideways to the right while grappling. Step into the cargo area
that the crate was blocking and pick up the --RANDOM AMMO-- from it.
[ ] --"Infected Soldiers" database file--[ ] Random Ammo
Run back around the corner then approach the other side where more kinesis
symbol crates are. Walk to the far right along that side then turn to face the
crate that blocks the way. Grapple the kinesis crate and move directly into
the area that you just stepped through. Grapple the left crate that was behind
the one you just moved and push it back further into the room. Turn to the
right and find the next kinesis crate then slide it over to the right. This
path will move Isaac into the back of the cargo room.
The enemy that has been keeping ahead of Isaac, the Stasis Slasher, will be
waiting at the end of the corridor of crates further ahead.
These things move
incredibly fast when they run toward Isaac. Shoot him with stasis whenever he
moves in front of Isaac then blast him like any other enemy. You have to be
careful while attempting to shoot them with stasis since they will sometimes
duck whenever they dash toward Isaac and the stasis shot will fly over their
head. The Force Gun's primary fire works very well against these type of
enemies without the need to use stasis, simply wait for one to run up to Isaac
then shoot it with a primary fire blast and it will most likely loose some
appendages and get knocked back. Run up to it and finish it off with more
These enemies are just as deadly without a leg, so shooting off their legs
won't do that much to cripple them. Shoot off their appendages as you normally
would with a Slasher to kill them. They are tougher than the normal Slashers
but the main hard part about them is how fast they are! Two more Stasis
Slashers will attack after the first one is defeated.
Move on ahead down the corridor of crates then take the first left. Grapple
the crate in the left corridor with kinesis then push it directly ahead. Run
into the left corridor then smash the --ITEM BOX-- and take the --RANDOM AMMO--
from inside.
[ ] Random Ammo[ ] Item Box
Look out for the reverse gravity plates up ahead. A Stasis Slasher will move
out a vent up ahead then attack. Approach the unlocked door off to the left
and use kinesis to move the small crates in front of it then enter the next
== Torpedo Room ==
You'll need to grapple the crates blocking the doorway and toss them out into
the room ahead. Look off to the left and open the first --ITEM CHEST--.
You'll need to grapple the small crates directly ahead and shoot them out of
the way to get to the --ITEM BOX-- and --ITEM CHEST-- on the left side of the
opposite side of the room. Don't mess with the stacked crates near the vent
across from the entrance side or a Stasis Slasher will bust through the vent
and attack.
[ ] Item Chest[ ] Item Chest[ ] Item Box
There is no point in moving the power unit away from the power plug. Move down
the walkway in the middle of the room. The --HAMMOND ASSISTANCE-- database
file will be received on the other side of the room. There is an
--ITEM CHEST-- containing a --GOLD SEMICONDUCTOR-- to the right as Isaac steps
off the walkway. Enter the next room.
[ ] --"Hammond Assistance" database file--[ ] Item Box
[ ] Gold
== Port Corridors ==
Take note of the --ITEM CHEST-- near the right wall as Isaac moves toward the
!!SAVE STATION!! up ahead. Mutilate all the soldier bodies in this hallway
before you mess with the closest door up ahead (it's an elevator door). The
furthest door is blocked from the other end. When Isaac attempts to open the
elevator door, the power unit beside it will explode. Infectors will attack
right after the power unit is destroyed, but if you stomped the bodies then
they will have nothing to infect, so take out a gun and defeat them. If the
soldier bodies are still there then you better blast the Infectors before they
use the bodies as a host since they will make Stasis Slashers out of the dead
Run down the hall and grapple the power unit that powers the door at the other
end then pull it out of the power plug. Bring it back over to the empty power
plug beside the elevator door then plug it in to restore power to the elevator.
Enter the elevator and take it up. On the way up the elevator shaft, the --DR.
KYNE-- database file will be received.
[ ] --"Dr. Kyne" database file--
== Engine Corridors ==
You'll likely notice a man yelling in pain here.
Look on the wall in the
middle of the burning debris directly ahead. A crew member is trapped on the
ceiling because of the reverse gravity plate below him. You can't shoot him to
end his suffering... then use kinesis to pull his dead body toward Isaac and
stomp his remains into the floor. The game won't allow it, so let's continue
down the hall to the side.
Step off into the right hall along the way to the next door and open the --ITEM
CHEST-- and --ITEM CONTAINER-- in the room with the dead soldier. Walk toward
the burning debris and grab the --RANDOM AMMO-- to the left behind it with
[ ] Item Chest[ ] Item Container[ ] Random Ammo
== Armory ==
This room can be dangerous at first. A Divider will dash at Isaac
from the
area across from the door and a few Exploders will walk toward the door from
off to the right. Be careful about entering this room. You might want to
shoot the Divider with stasis then run to the area where he comes from and turn
back around to deal with the Exploders as they appear from the side. You can
also step back into the prior hallway.
Grab the --POWER NODE-- and take note of the --ITEM CHEST-- and --ITEM BOX--
off to the right. There is an !!UPGRADE BENCH!! off to the side. Two
Exploders will move through the open vent, near the door that Isaac entered
this room from, when Isaac walks further to the right, so be ready for them.
Pick up the --EMERGENCY-- database file from the shooting gallery area directly
across from the entrance door then open the nearby --ITEM LOCKER--.
[ ] Item Chest[ ] Item Box[ ] Item Locker,
[ ] --"Emergency" database file--
+ + Shooting Gallery Minigame + +
You may want to try out the shooting range minigame. Once Isaac begins the
game he will have infinite ammo with whatever weapon he has equipped. Shoot
the red figures and avoid shooting the blue figures until the max score is
reached as presented on the back board. Either the Plasma Cutter or the Pulse
Rifle is the best weapon to use here. There are five rounds altogether and
Isaac will receive an item after every successfully completed round - it
appears on the counter to the left of the controls. If you mess up during a
round, simply back away to cancel the game then move forward and restart.
Here is the required score and items won:
Round 1 - 1000 score
Reward: Random Ammo
Round 2 - 1200 score
Reward: Random Ammo
Round 3 - 1400 score
Reward: Medium Med Pack
Round 4 - 1600 score
Reward: Ruby Semiconductor
Round 5 - 2000 score
Reward: Power Node
Rounds 4 and 5 get very fast so keep your Plasma Cutter set to horizontal and
listen for the confirmation for each shot. If you fail to hit one back up then
move forward to try again. Don't pay attention to the scoreboard when you get
to Rounds 4 and 5 or it will be just enough to distract you away from a target.
Isaac can sometimes be the worst part of this entire game since the he covers
the left side of the camera if you stand in the middle, which is really the
best place to stand. Keep your gun aimed toward the middle then shoot a target
and return aim to the middle then wait for the next. On round 5, you have to
simply keep aiming and shooting.
Step into the next room outside when you're ready.
== Armory Corridors ==
A soldier will die from a leg wound as Isaac enters this area.
Poor fellow.
You can mutilate his dead body if you want. Be sure to recharge your stasis at
the stasis recharger if needed. Enter the Infirmary.
== Infirmary ==
A Stasis Slasher will show Isaac what happens whenever a living thing runs
through the lasers that revolve around the middle of the room.
It isn't pretty
and the same fate will befall Isaac if he happens to touch them. The robotic
arm controls the lasers and it can be shot with stasis. Wait for the arm to
point the lasers to the right then hit it with stasis and run over to the left
then pick up the --LARGE MED PACK SCHEMATIC-- and open the --ITEM CHEST-- and
run to the other side of the room very quickly.
A Stasis Slasher will attack from the other side while Isaac runs, but just run
by him or stun him with stasis quickly then run by him. He might get caught up
in the lasers himself. Open the two --ITEM CHESTS-- on the other side of the
room then enter the Barracks Corridors.
[ ] Large Med Pack Schematic[ ] Item Chest[ ] Item Chest[ ] Item Chest
== Barracks Corridors ==
You'll hear a yell in here from an Exploder, so run down the hall to the right.
A Stasis Slasher will be the first to come after Isaac, so shoot him with
stasis then defeat him. Exploders will move down the hall toward Isaac next,
so shoot them with the Plasma Cutter or Pulse Rifle from a distance. An
Exploder will fall near Isaac but he should be far enough away to shoot it
safely. A few more Exploders will appear down the hall also. Grab the --POWER
NODE-- and use kinesis to pull the --RANDOM AMMO-- from the right side of the
burning debris toward Isaac.
[ ] Power Node[ ] Random Ammo
Continue back down the hall then move down the end of the hall across from the
door that Isaac entered this area from. Open the --ITEM CHEST-- to the right
then move further ahead and open the other --ITEM CHEST-- to the left. Grab
the --RANDOM AMMO-- and --SMALL MEDPACK-- from in front of the debris and save
your game at the !!SAVE STATION!! on the right wall. You'll probably notice an
Exploder moving to the left behind the debris but it will step out of sight
quickly. Enter the Barracks.
== Barracks ==
A quarantine will initiate once you move further into this room. Run back to a
corner and stay there.
Stay in the left corner of this room so enemies might
possibly walk across the reverse gravity plating up ahead. A Pregnant will be
the first to appear in the back but it shouldn't come toward Isaac if you step
back quickly enough. Lurkers and a Stasis Slashers will be the first to attack
followed by Stingers. Another Stasis Slasher will attack eventually. There is
really too many enemies in this room to keep up with any type of pattern, so
just know that you'll face plenty of Stasis Slashers, Stingers, Lurkers,
Exploders, and a Pregnant.
If you approach this room cautiously by staying in a corner near the back then
it is not as bad as it can be. If you dart into the middle of the room then
prepare for an onslaught! Use the Plasma Cutter or Pulse Rifle to shoot at
distant Exploders, use the Force Gun or Line Gun for Stasis Slashers, use the
Flamethrower or Force Gun for Lurkers then fight off the Stingers with the
Plasma Cutter or Line Gun. If you stay away from the other end of the room
then the Pregnant and Exploders from that end will most likely not even move
toward Isaac. Once the lockdown has been lifted, all enemies in the room are
Check the second opening in the right wall and grab the --LARGE MED PACK-- from
inside. You'll find plenty of --ITEM CONTAINERS-- on the large pillars in the
middle of the room - there are seven unlocked containers. At the far end of
the hall is a !!STORE!! on the back wall and the --ORDERS-- database file along
the floor. Enter the Engine Corridors through the door at the back end of the
[ ] Large Med Pack
[ ] Item Container
[ ] Item Container
[ ] Item
Container[ ] Item Container[ ] Item Container[ ] Item Container
[ ] Item
Container[ ] --"Orders" database file--
== Engine Corridors ==
Grab the --RANDOM AMMO-- from near the debris. We're now on the other side of
the floor where Isaac exited the lift. The guy that is trapped by a reverse
gravity plate is still held up against the ceiling and it looks as if he is
still ticking - he's got some determination there.
[ ] Random Ammo
Move toward the !!SAVE STATION!! on the wall directly ahead and save your game.
Follow the hall as it turns to the left and open the --ITEM CHEST-- near the
end then open the next door.
[ ] Item Chest
== Engine Hall ==
A man will step into view then run to the right further ahead - he actually
opens a door.
Walk down the hall and open the two --ITEM CHESTS-- then stomp
the --ITEM BOX--. There are three more --ITEM CHESTS-- near the locked door at
the other end of the hall. Open the large door to the right that the man ran
[ ] Item Chest[ ] Item Chest[ ] Item Chest[ ] Item Chest[ ] Item Chest,
[ ] Item Box
== Engine Room 1F ==
Find the two --ITEM CONTAINERS-- along the sidewalls then open them. Take the
lift to the top floor.
[ ] Item Container[ ] Item Container
== Engine Room 2F ==
The flames coming from the burners in the very back of this room will burn
Isaac and damage him quite a bit if he steps out into the flames - thankfully
they won't kill him instantly. Isaac can stand behind the metal cylinders with
the kinesis symbol on them to avoid the flames. He can grab these cylinders
with kinesis and move them toward the back of this room to get further into the
room unharmed.
There are three purple fuses along the middle portion of each side of this room
for a total of six. The fuses must all be destroyed to shut off the burners in
the back of this room. Grapple either cylinder and push it toward the burners
then shoot or punch the purple fuses along the metal columns on each side. On
the right side they are on the left columns and on the left side they are on
the right columns.
It is best to only move the metal cylinders when the flames stop. If Isaac is
too far away from the metal cylinder when the flames start then he will get
burned and this can easily be done while using kinesis while the flames are
surrounding Isaac. You will have to move the cylinders to the right or left
sometimes to avoid the pipes near the metal columns.
Once all six fuses are destroyed, the flames will stop. Collect the
++SINGULARITY CORE++ from the device in the middle of the back portion of the
Objective Complete:
[X] Find the Singularity Core
Once the core has been collected the --SINGULARITY CORE FOUND-- database file
will play and Isaac will receive the next objective.
[ ] ++SINGULARITY CORE++[ ] --"Singularity Core Found" database file--
Objective Added:
[ ] Go to the Crew Deck
I've got the Singularity Core now. Removing it may cause some bad side
effects to the Valor's power system, so I should get out of here quickly.
I'm heading to the Crew Deck now, in hopes of repairing the shuttle docked
The entire ship will now start to malfunction with short explosions throughout
the rooms now that the Singularity Core has been removed. The explosions for
the moment won't hurt Isaac and there is no hidden timer for getting out of the
ship. Find the corridor off to the right and move down it. Open the three
--ITEM CHESTS-- to the right at the first turn then continue to the left. An
explosion will engulf the next three --ITEM CHEST-- in flames but thankfully
the flames cannot hurt Isaac. Open the door to the left.
[ ] Item Chest[ ] Item Chest[ ] Item Chest[ ] Item Chest[ ] Item Chest,
[ ] Item Chest
Open the --ITEM CONTAINER-- on the wall to the left after stepping through the
doorway. As Isaac reaches the glass in the back, someone will step into view
from the other side. Ah, it's Hammond. What's up, boss?
[ ] Item Container
A Dark Brute will attack as it steps through the glass from the other side. Be
sure to shoot it with stasis then get in the back of it like all the rest. The
Contact Beam is the best way to defeat it quickly. If you don't have stasis
then shoot it with the Contact Beam from the front and it will eventually hunch
over to where you can get in some good back shots. The Line Gun works just as
well also. It will drop a --DIAMOND SEMICONDUCTOR-- when it is defeated. The
--HAMMOND'S DEATH-- database file will be received once the Brute is defeated.
[ ] Diamond Semiconductor[ ] -- "Hammond's Death" database file--
Be sure to stomp the hell out of that Dark Brute body. Hammond's body can be
mutilated also, but you don't really want to do that, do you? Don't let sudden
memories of the ADS cannon accomplishment in Chapter 4 cloud your mind, Mr.
Clarke! Open the large door ahead then step back into the Engine Hall.
The door to the right is now unlocked so move through it. An explosion will
happen near the door that Isaac just went through that will set it ablaze.
warning message will soon sound over the intercom. Avoid all the flames in the
next few areas along with this one or Isaac will take damage.
== Corridors ==
Move down the hall to the left. Open the lone --ITEM CHEST-- off to the right
along the way. Use kinesis to grab the power unit along the way and then plug
it into the nearby power plug to the right of the elevator door to unlock it.
Open the --ITEM CONTAINER-- on the wall to the right then step inside of the
elevator. Big explosions will trigger in the hall outside but Isaac is safe in
the elevator. Take it down.
[ ] Item Chest[ ] Item Container
Rush directly ahead and avoid the flames and the reverse gravity panel then
save your game at the !!SAVE STATION!!.
Walk back to the elevator door and
find the exit to the Valor off to the side. Exit the ship by stepping through
the airlock and then the next door ahead.
Isaac will once again enter the vacuumed zero gravity room outside. Leap to
the floor below as the USM Valor continues to explode.
Use the oxygen
rechargers surrounding the middle doorway in the center of the room if needed.
Find the walkway with the door on the opposite side along the wall then open
the door and step inside the airlock. Allow air to restore then open the next
== Tram Station ==
A Stasis Slasher will step out from the left corner up ahead, so take out the
Force Gun and blast our unwelcome guest. Step back into the Tram at the end of
the hall then interact with the controls to end this chapter.


text:
CMSText, a mobile marketing company founded in Lexington, announced plans Thursday to add 135 workers.
"With the growth that we've had, we're in a position now to expand our operations," said founder Conrad Carney, who played football at the University of Kentucky in the 1980s.
Carney built CMSText from its roots with text message marketing utilized by industries including restaurants, and now the firm has designed a new platform that he hopes to launch in the first quarter of 2012.
Called Xooker, he said the platform is a step beyond social networks like Facebook and Twitter and is more akin to products like Groupon and Living Social in which people sign up for marketing with deals and so forth.
"If you like Groupon, it will be Groupon on steroids," he said, explaining, though, that Xooker encompasses lots more.
"It will also have mobile gaming involved," Carney said. "It's a complete mobile, social new media platform.
"The Groupon-esque aspect of it is only one small portion of the platform."
Carney said the company has already hired 20 additional employees as it built the platform and will be looking for more than a hundred more starting next year as it rolls out the platform in various markets. The jobs will range from computer programmers to marketers.
"A lot of the hiring depends on the markets themselves and how quickly we're opening those," Carney said. "We hope to be adding sooner rather than later."
"We feel that in the first two years, we'll be hitting those numbers," he added of the 135 jobs total.
As they become available, the jobs will be advertised on CMSText.com, Carney said.
The Kentucky Economic Development Finance Authority on Thursday gave preliminary approval for CMSText to receive $2.5 million in state tax incentives if it indeed makes the hires. The state noted in documents that the expansion is expected to cost CMSText roughly $4.4 million.
In planning the expansion and securing the tax incentives, the firm worked with the state Cabinet for Economic Development and the Bluegrass Business Development Partnership, which includes representatives of Lexington's government, Commerce Lexington and the University of Kentucky.
"We're delighted to have been a part of the process working with Conrad Carney and his team," said Gina Greathouse, Commerce Lexington's senior vice president for economic development. "I think it speaks highly of Lexington's economy and our employment base that a technology company has started up in Lexington and is choosing to grow here."
Since graduating from UK, Carney has witnessed the evolution of mobile technology first hand in Lexington. He went to work initially for a cell-phone company and recalls how it celebrated its 40th customer in 1987.
When Carney founded CMSText in August 2007, his focus was primarily on text message marketing. The company specialized in restaurant marketing, having created an easy-to-use tool that allowed restaurateurs to text offers and other information to customers who opted in.
The company grew to seven employees in 2009, as Carney brought on sales managers in regional areas.
Building off that work, CMSText was named the exclusive mobile text marketing partner of the National Restaurant Association. Carney then expanded the company to focus on the health care industry, as doctors' offices began to utilize the text message marketing to remind customers of appointments or to take pills. Now it's evolved again, he said, with Xooker.
"It's about utilizing technology to market more efficiently and, more importantly, less expensive for businesses."


text:
Kiergan is a character concept created by Kristof.
Kiergan is a character concept who belongs to Kristof's Dark Saga.
Kiergan and his foster brother Vogar were elite members of Dakkon's army known as the Black Guard. Keirgan blamed a failed campaign on Vogar who was almost killed by Dakkon. Later in the insurrection against Dakkon, Vogar faced and killed Kiergan.


text:
No Recent Price Change
2841 Parkers Landing Road
Mount Pleasant, SC 29466
- Vacant Land
- Active
Agent Remarks
Beautiful water front home site in sought-after Parkers Landing section of Rivertowne Country Club. Build your dream home in this private setting on a quiet cul-de-sac with beautiful marsh and river views. Enjoy golf at Rivertowne Country Club's Arnold Palmer Signature Course then meet friends at the club for dinner. Additional amenities include neighborhood pool, tennis courts walking trails and play park all conveniently located to beaches, downtown Charleston, world class dining and shopping.
Lot Info
Acres: 0
Lot Desc: Cul-de-sac, Marshfront, Marshview, River Access, Riverview, Tidal Creek, Waterfront - Shallow, Waterfront - Deep
Misc Features
Auction: No
Property Features
Subsection: Parkers Landing
Uses: Residential
Property Type: C
Property Info
Zoning: Residential
School Elem: Jennie Moore
School Middle: Laing
School High: Wando
Courtesy of listing office Carolina One Real Estate


text:
Counselor February 13, 2017
Q&A: John Mackey, Whole Foods
Whole Foods Co-Founder John Mackey accelerated the organic industry and brought healthy food to millions of people.
Two decades ago, sales of organic products in the U.S. totaled $3.6 billion. Today, it's over $45 billion. And perhaps no person is more responsible for that staggering growth than John Mackey. The CEO and co-founder of Whole Foods started with a single store in 1980 and grew the natural food grocer into a titan of healthy living, with over 460 locations today and nearly $16 billion in revenue. Organic food is now available in grocery stores and markets around the country due to the demand that Mackey and Whole Foods tapped into.
But Mackey espouses something greater. His book Conscious Capitalism urges businesses to strive for a higher purpose instead of just aiming to make money. An Austin, TX, native and the keynote speaker at ASI Show Dallas, Mackey shares with Counselor some of the secrets that have made Whole Foods a two-decade mainstay on Fortune's Best Places to Work list, and why he's never regretted cutting his salary to $1 a year.
Counselor: What is conscious capitalism?
John Mackey: We identify four pillars with conscious capitalism. First, every business has a potential for a higher purpose that goes beyond just making profits. Second, the business should be managed on behalf of all of the interdependent stakeholders: customers, employees, suppliers, investors, communities. Third, you should have a different style of leadership than traditionally has been the case. We believe that it's more of a servant leadership. The leaders serve the higher purpose of the business; they serve the stakeholders. And fourth, you need to create a culture that really helps people who work for the business to flourish, grow and evolve as human beings and leaders.
Counselor: What are some keys to building that type of corporate culture?
JM: There are a number of factors. The first one, of course, is that the culture should be in some sense an extension of the purpose of the business. Why does the business exist, and what kind of value is it creating in the world? There's not one perfect culture. It depends on what kind of business you're in and what its purpose and value creation is.
Secondly, it's essential that the leadership walks the talk. You can't just have core values, a higher purpose, principal, things like that, if the leadership doesn't really believe it and doesn't lead by example. People will pay a lot more attention to what you do and how you live than what you say.
Generally, a successful culture is one that is going to have greater transparency in it. It's going to encourage authenticity. It's going to be one where there's a greater sense of care and love in the organization, one that has integrity and tries to do the right thing. It's fundamentally honest and trustworthy. The basic virtues that go into making up a good human being also are the virtues that make up a good organizational culture.
Counselor: How have you been able to develop that culture at Whole Foods?
JM: I would say when we started out we didn't do it very consciously. We wanted to have a business, and our initial higher purpose at Whole Foods was pretty simple, which was just to sell healthy food to people, earn a living for ourselves and have some fun doing it. And those are still in the DNA of the company. But of course, the purpose has gotten deeper as we've gotten bigger and it's more encompassing.
Then we also wanted to create a culture that we ourselves would want to work in. We thought about what we didn't like. We wanted to make sure we didn't replicate that. We didn't want to attract leaders who were primarily into power over others, or who were more interested in their own compensation. So we had to establish the basic cultural elements of transparency, authenticity.
For example when I say transparency, at Whole Foods Markets what everybody gets paid is in the public realm. We don't keep that hidden away. We want to empower our team members about the financial situation – not only of the company, but of their particular work unit so they can see how they're doing. They want to link their own bonuses into how well the company and business unit are doing. So we reward people both at the micro level and the store level.
Frankly, most cultures, they're sort of taken for granted. It's one of my most important jobs to look at the culture. To use a metaphor, it's like your garden, you've got to root out the weeds and bad elements, and you've got to nurture the virtues and plants that you want to grow.
Counselor: Ten years ago you decided to cut your salary to $1 a year. Have you ever regretted that decision?
JM: I never personally regretted it. I'd have to say my wife regretted it back in 2008 when everything was crashing down. She was like, 'why the heck did you cut your salary to $1? You should get your salary back. We kind of need the money,' and I said, 'I can't do that. I would be a laughingstock. We're just gonna have to struggle through this and we'll get to the other side of it.' So I've never had any regrets. I'm actually glad I did it, because it makes it very clear to everybody at the company that I'm not in it to maximize my own personal gains, but to serve the company to help it reach its highest potential. It takes self-interest off the table when people want to ask questions about that. I'm not saying anybody else should necessarily do it, but it worked out well for me and Whole Foods.
Counselor: I wanted to ask you about your dad, who helped you start the company. Did you approach him with this idea of a natural and organic supermarket?
JM: I did. I was very young. I think I was 24 when I pitched my dad about starting a store. I had absolutely no background in business whatsoever. I studied philosophy and the humanities when I was in school and actually never took a degree. I just took electives. My dad had been an accounting professor at Rice University and then he'd gone into business, and so it was really good because he and I got really close and worked together very effectively for about 16 years.
But he was initially pretty skeptical. He said "Are you sure that's what you want to do? You want to be a grocer? Organic foods, what's that?" This is back when almost nobody knew what organic meant and it was all seen as very weird stuff. But I was so enthusiastic about it. I was so excited about it. I found my life's passion. My dad went along with it. He loaned me $10,000 to put into the business, and we capitalized the business initially with $45,000 from friends and family like a lot of startup businesses. And we managed to lose half that money in the first year, I might add, before I learned enough to turn it around and make the business profitable in year two.
Counselor: What is the best advice you have for entrepreneurs?
JM: Ultimately, it's about value creation. What kind of value can you create in the world that people want? Is your idea an idea that other people are gonna embrace? And if so, you gotta go for it. Most entrepreneurs I've known are very passionate people that usually have a dream they're trying to realize. So my biggest advice is encouragement to do it.
I'll tell you a story. Oftentimes when I talk at business schools I will take a quick poll. These are mostly MBA students getting their masters in business administration. And I'll ask the class how many of them plan to start their own business some day. And about 75%-90% of the people raise their hand. And then I follow-up and ask how many of them plan to do that immediately after they get their MBA. Most of the hands go down.
Usually only a few stay up. And then I'll tell them, the people that still have their hands up, they're the ones that are going to create their own businesses. And the rest of them are probably kidding themselves. Because they're gonna go into consulting, or they're gonna get hired by Goldman Sachs or investment banks or consulting firms. And they're gonna make big salaries and they're probably not gonna go start a business someday because they're just gonna go down a different track.
So my advice if you're an entrepreneur is get on with it. Stop wasting time and get on and build your business. Create your dreams. Follow your heart and live a life with passion. It may not work out for you, but if you're true to yourself, it probably will.