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Recent collaborative efforts have subclassified malignant glioblastomas into 4 clinical relevant subtypes based on their signature genetic lesions. Platelet-derived growth factor receptor α (PDGFRA) overexpression is concomitant with a loss of cyclin-dependent kinase inhibitor 2A (CDKN2A) locus (encoding P16INK4A and P14ARF) in a large number of tumors within one subtype of glioblastomas. Here we report that activation of PDGFRα conferred tumorigenicity to Ink4a/Arf-deficient mouse astrocytes and human glioma cells in the brain. Restoration of p16INK4a but not p19ARF suppressed PDGFRα-promoted glioma formation. Mechanistically, abrogation of signaling modules in PDGFRα that lost capacity to bind to SHP-2 or PI3K significantly diminished PDGFRα-promoted tumorigenesis. Furthermore, inhibition of SHP-2 by shRNAs or pharmacological inhibitors disrupted the interaction of PI3K with PDGFRα, suppressed downstream AKT/mTOR activation, and impaired tumorigenesis of Ink4a/Arf-null cells, whereas expression of an activated PI3K mutant rescued the effect of SHP-2 inhibition on tumorigenicity. PDGFRα and PDGF-A are co-expressed in clinical glioblastoma specimens, and such co-expression is linked with activation of SHP-2/AKT/mTOR signaling. Together, our data suggest that in glioblastomas with Ink4a/Arf deficiency, overexpressed PDGFRα promotes tumorigenesis through the PI3K/AKT/mTOR-mediated pathway regulated by SHP-2 activity. These findings functionally validate the genomic analysis of glioblastomas and identify SHP-2 as a potential target for treatment of glioblastomas.
Human gliomas account for the most common and malignant tumors in the CNS (1). Despite intensive treatments including maximal surgical resection, combined with radiotherapy and concurrent or adjuvant chemotherapies, median survival time of patients with high-grade glioblastoma multiforme (GBM) remains 14–16 months after diagnosis (1, 2). Recently, coordinated genomic analyses of a large cohort of clinical GBM specimens identified frequent co-alterations of genes in 3 core pathways — the P53, retinoblastoma (RB), and receptor tyrosine kinase (RTK) pathways — that are crucial in gliomagenesis (3). Notably, the gene encoding platelet-derived growth factor receptor α (PDGFRα) is amplified in approximately 13% of total GBMs analyzed, and deletion of the tumor suppressor cyclin-dependent kinase inhibitor 2A (CDKN2A) locus is frequently found in these PDGFRα-amplified GBMs (3, 4). However, to our knowledge, studies have not been conducted to determine whether these genetic aberrations act in concert to promote gliomagenesis, or to determine the underlying mechanisms of PDGFRα-stimulated tumorigenesis.
The homozygous deletion of the CDKN2A locus at chromosome 9p21, which eliminates both INK4A and ARF genes (encoding P16INK4A and P14ARF, respectively), concomitantly deregulates 2 of the major tumor suppressor pathways, the RB and P53 pathways (5). Mice lacking the Cdkn2a locus develop spontaneous tumors and are more prone to carcinogenesis (6). Whereas no spontaneous tumors are found in the brain of Ink4a/Arf-deficient mice, astrocytes and neural stem cells (NSCs) from these mice form high-grade gliomas in the brain upon EGFR activation (7). Additionally, K-Ras activation has been shown to cooperate with loss of Ink4a and Arf in mouse astrocytes (mAsts) or neural progenitor cells to generate GBMs (8).
PDGFRα is a RTK that elicits a variety of biological activities such as cell proliferation and migration via stimulation by its ligand dimers, PDGF-AA, -AB, -BB, -CC, and -DD. Activated PDGFRα associates via the autophosphorylated tyrosine (p-Y) residues at its cytoplasmic domain to various downstream SH2 domain–containing signaling molecules, including SRC family kinases (SFKs), phosphotyrosine phosphatase SHP-2, PI3K, and PLCγ (9, 10). Mice deficient in PDGFRα or PDGF-A or engineered to separately express PDGFRα with mutations of the individual p-Y sites show alterations in cellular behavior and embryo development (9, 11). In particular, PI3K has been identified as the major effector of PDGFRα signaling in vitro and in vivo (10, 12, 13). SFKs and PLCγ contribute to some but not all PDGFRα functions (10, 12–14), whereas SHP-2 is not required for cell survival during Xenopus embryogenesis (12). However, contributions of each of these signaling modules to glioma formation have not to our knowledge been demonstrated.
Activation of PDGFRα signaling has been observed in human gliomas. In clinical glioma specimens, PDGFRα and PDGF-A are overexpressed in tumor cells, while PDGFRβ is only expressed in endothelial and peri-endothelial compartments (15). PDGF-B, which binds to both PDGFRα and PDGFRβ, is an oncoprotein that causes glioma formation in the brain (16). Loss of Ink4a/Arf in mAsts enhances PDGF-B–initiated gliomagenesis and tumor progression in the brain (16). Specific activation of PDGFRα signaling by infusion of PDGF-A proteins, which only bind to PDGFRα in PDGFRα-positive type B NSCs in the subventricular zone (SVZ), leads to glioma-like growth of these cells in adult brain (17). However, how the activation of PDGFRα signaling causes glioma formation and whether co-alteration of tumor suppressor pathways is required in PDGFRα-mediated gliomagenesis have not been directly shown. In this study, we determined the synergistic impact of PDGFRA overexpression and INK4A/ARF deletion on gliomagenesis in mAsts and human glioma cells. We then used genetic and pharmacological approaches targeting individual downstream signaling enzymes to examine which specific signaling pathway(s) emanating from PDGFRα are critical in tumorigenesis. Furthermore, we confirmed our observations in clinical glioma specimens that co-overexpress PDGFRα and PDGF-A.
To determine whether activation of PDGFRα signaling in Ink4a/Arf–/– mAsts leads to gliomagenesis in the brain, we overexpressed PDGFRα and/or its ligand PDGF-A chain in a retroviral vector containing an IRES-GFP in Ink4a/Arf–/– mAsts (Figure (Figure1A).1A). The impact of PDGFRα/PDGF-A overexpression on the growth and survival of these cells in vitro was moderate (Supplemental Figure 1, A and B; supplemental material available online with this article; doi: 10.1172/JCI43690DS1). However, when these cells were separately transplanted into the flanks of mice, significant s.c. tumor growth was evident in mice that separately received Ink4a/Arf–/– mAsts expressing PDGFRα, PDGF-A, or PDGFRα/PDGF-A (with the receptor and ligand co-expressed in the same cell to create an autocrine signaling), whereas minimal or no tumor formation was seen in control mice that received Ink4a/Arf–/– mAsts expressing GFP (Figure (Figure1B).1B). To determine tumorigenicity of these cells in the brain, various mAsts were separately implanted into the brain of mice. Notably, mice that received GFP mAsts did not show active tumor growth up to 42 days after implantation, while PDGFRα, PDGF-A, and PDGFRα/PDGF-A mAsts started to form tumors in the brain as early as 8–11 days and the majority of tumors reached a volume of approximately 25 to 30 mm3 in 25–35 days. Moreover, mice that separately received PDGFRα- or PDGF-A–expressing mAsts survived up to 2 months after implantation. On the other hand, all mice that received autocrine PDGFRα/PDGF-A–expressing mAsts developed large and invasive tumors by 20 days, with an average survival time of 25 days after implantation. As shown in Figure Figure1,1, C–L, significantly larger and highly invasive gliomas formed in the brains of mice that received Ink4a/Arf–/– mAsts overexpressing PDGFRα, PDGF-A, or PDGFRα/PDGF-A, whereas only small tumor lesions were found in the brain of mice that received control mAsts. Significantly, an approximately 10-fold increase in the cell proliferation index was found in gliomas derived from PDGFRα-activated mAsts compared with control tumors (Figure (Figure1,1, M–R), whereas an approximately 10-fold decrease of cell apoptosis was seen in PDGFRα/PDGF-A–expressing tumors (Figure (Figure1S).1S). Of note, in established s.c. or brain tumors, exogenous expression of PDGFRα or PDGF-A was maintained at the end of the experiments (Supplemental Figure 1, C–K, and data not shown). Taken together, these results indicate that expression of PDGFRα and/or PDGF-A confers tumorigenicity to Ink4a/Arf-deficient mAsts in the brain.
To further characterize the tumors derived from Ink4a/Arf–/– PDGFRα mAsts, we examined molecular markers of various cell lineages in the CNS development. As shown in Figure Figure2,2, tumors derived from PDGFRα-expressing mAsts were highly positive for the neural progenitor marker nestin, which was distributed along the processes of individual cells (Figure (Figure2,2, A and B). As expected, most of tumor cells showed expression of the progenitor/mature astrocyte marker GFAP (Figure (Figure2,2, C and D) but were negative for the neuronal marker class III β-tubulin (TUJ1) (Figure (Figure2,2, E and F). Significantly, NG2, an oligodendrocyte progenitor cell marker (18), was expressed in a population of tumor cells that were actively invading the surrounding brain parenchyma, whereas the core of the tumor mass showed relatively low NG2 expression (Figure (Figure2,2, G and H). In contrast, control tumors derived from Ink4a/Arf–/– GFP mAsts lacked nestin or NG2 expression (data not shown). In addition, tumors derived from Ink4a/Arf–/– PDGFRα mAsts showed negative staining for oligodendrocyte type 2 astrocyte progenitor marker A2B5 and an oligodendrocyte marker CNPase (data not shown). Notably, brain tumors derived from Ink4a/Arf–/– PDGFRα/PDGF-A mAsts (data not shown) displayed similar IHC features to tumors derived from Ink4a/Arf–/– PDGFRα mAsts, suggesting that similar dedifferentiation events also occurred in autocrine PDGFRα/PDGF-A–co-expressing tumors.
Next, we used 4 human glioma cell lines that were either INK4A/ARF null (LN444 and LN443) or WT INK4A/ARF (LN-Z308 and LN319; Figure Figure3A)3A) (19) and exogenously expressed PDGF-A in these glioma cells that express endogenous PDGFRα (Figure (Figure3B).3B). In vitro, expression of PDGF-A in INK4A/ARF-null LN444 cells significantly enhanced their capacity of anchorage-independent growth in soft agar, whereas a minimal effect was seen in WT INK4A/ARF LN-Z308 and LN319 cells (Figure (Figure3C).3C). When various glioma cells were separately implanted into the brain or the flanks of mice, PDGF-A expression markedly enhanced tumor growth and invasion of INK4A/ARF-null LN444 and LN443 glioma cells, while minimal impact of PDGF-A expression on tumorigenesis or invasion was seen in WT INK4A/ARF LN-Z308 and LN319 cells in both anatomic sites (Figure (Figure3,3, D–K, and Supplemental Figure 2). Mice received LN444/PDGF-A cells in the brain had tumor onset as early as 35 days, while mice with LN443/PDGF-A cells developed invasive intracranial tumor 25–30 days after implantation. In contrast, LN444 and LN443 parental cells only formed small tumor lesions in the brain up to 2–3 months following implantation. Mice that received LN444/PDGF-A cells survived for 75–80 days, while most of the mice that received LN443/PDGF-A cells lived up to 3 months after implantation. On the other hand, no significant brain tumor growth was found 2–3 months after implantation in mice that received LN-Z308/PDGF-A or LN319/PDGF-A cells. Thus, consistent with our findings in the Ink4a/Arf–/– mAsts, these data indicate that PDGFRα/PDGF-A signaling enhances in vivo tumor growth and invasion of human glioma cells deficient in INK4A/ARF.
To investigate whether re-expression p16INK4a or p19ARF is able to abrogate the enhanced tumorigenicity in Ink4a/Arf–/– mAsts, we separately expressed these 2 tumor suppressors in mAsts (Figure (Figure4A).4A). Surprisingly, re-expression of p16INK4a but not p19ARF alone in Ink4a/Arf–/– mAsts suppressed soft agar growth of PDGFRα-expressing mAsts stimulated by PDGF-A (Figure (Figure4B).4B). Consistently, enforced restoration of p16INK4a in Ink4a/Arf–/– mAsts significantly inhibited tumorigenesis of PDGFRα/PDGF-A–overexpressing mAsts in the mouse brain (Figure (Figure4C).4C). To further demonstrate the cooperative effect of PDGFRα activation and loss of Ink4a on tumorigenesis, we knocked down endogenous p16INK4a using shRNAs targeting CDKN2A in WT INK4A/ARF LN319 cells. As shown in Figure Figure4D,4D, significant inhibition of P16INK4A without affecting P14ARF expression by 2 separate shRNAs in LN319 cells resulted in a marked increase in colony formation in soft agar. Since p16INK4a inhibits CDK4/6, which inactivates RB by phosphorylation, and since p19ARF (or P14ARF) targets MDM2, which suppresses p53 (5), we thus examined responses of modulating CDK4/6 and p53 in these cells. As shown in Figure Figure4E,4E, inhibition of phosphorylation of RB, the direct downstream target of CDK4/6 by CDK4/6 inhibitor PD0332991 (20), markedly reduced PDGF-A–stimulated cell growth of Ink4a/Arf-deficient mAsts and LN444 cells in soft agar, which suggests that the tumorigenic effect of PDGFRα signaling is dependent on CDK4/6 inactivation of RB proteins (p-RB). Since we did not observe any tumor-suppressing effect of p19ARF on Ink4a/Arf–/– PDGFRα-expressing mAsts (Figure (Figure4B),4B), we asked whether the downstream p53 protein was functional in these cells. When Ink4a/Arf–/– and Ink4a/Arf–/– p19ARF mAsts were treated with cisplatin (21), p53 expression was strongly induced in Ink4a/Arf–/– PDGFRα/p19ARF-expressing mAsts but only moderately induced in Ink4a/Arf–/– PDGFRα-expressing mAsts (Supplemental Figure 3A). The mAsts expressing p19ARF were more sensitive to cisplatin inhibition of cell survival than those without p19ARF expression, indicating that p19ARF-upregulated p53 in Ink4a/Arf–/– PDGFRα-expressing mAsts rendered sensitivity to cisplatin inhibition (Supplemental Figure 3B). Collectively, these data show that p16INK4a but not p19ARF suppresses the tumorigenesis promoted by PDGFRα/PDGF-A signaling, suggesting a cooperative effect of PDGFRα activation and p16INK4a inhibition during gliomagenesis. Additionally, p19ARF loss might be required for tumor survival in certain circumstances such as in the presence of DNA-damaging agents.
Previous studies using genetic and biochemical approaches have defined the roles of signaling molecules in PDGFRα-mediated cellular functions by specific tyrosine-to-phenylalanine (Y-to-F) mutations (Figure (Figure5A)5A) (10, 12, 13). To investigate the impact of these PDGFRα mutations on tumorigenesis, we separately expressed WT PDGFRα or various PDGFRα mutants in Ink4a/Arf–/– mAsts. The PDGFRα mutant R627 (PDGFRα-R627) that harbors a lysine-to-arginine (K-to-R) mutation was included as a "receptor kinase–dead" control. As shown in Figure Figure5B,5B, stimulation of WT PDGFRα by PDGF-A resulted in autophosphorylation of the receptor and promoted phosphorylation of downstream signaling molecules Erk1/2 and Akt, whereas there was an undetectable receptor tyrosine autophosphorylation in PDGFRα-R627 (Figure (Figure5B).5B). PDGFRα Y-to-F mutations at Y572 and Y574 (PDGFRα-F572/74; for SFK binding), Y1018 (PDGFRα-F1018; for PLCγ binding), and Y988 (PDGFRα-F988) did not result in a significant decrease in p-Akt and p-Erk levels in response to PDGF-A stimulation (Figure (Figure5B).5B). Moreover, the mutation at Y731 and Y742 (PDGFRα-F731/42; for PI3K binding) led to a marked decrease in PDGF-A–stimulated p-Akt and p-Erk1/2 levels (Figure (Figure5B).5B). In agreement with a previous report (22), p-Erk1/2 was markedly reduced in PDGF-A–stimulated mAsts expressing PDGFRα-F720 (Y-to-F mutation at Y720; for SHP-2 binding), compared with WT PDGFRα mAsts (Figure (Figure5B).5B). Interestingly, p-Akt levels were also attenuated in PDGFRα-F720 cells.
Next, we determined the impact of these Y-to-F mutations on PDGFRα-promoted cell transformation of Ink4a/Arf–/– mAsts in vitro. As shown in Figure Figure5C,5C, mAsts expressing WT PDGFRα and PDGFRα-F572/74, PDGFRα-F988, and PDGFRα-F1018 PDGFRα mutants showed similar capability of anchorage-independent growth in soft agar. In contrast, expression of PDGFRα-R627, PDGFRα-F731/42, or PDGFRα-F720 instead of WT PDGFRα significantly abrogated the capacity of mAsts to form colonies in soft agar (Figure (Figure5,5, C and D), indicating that PI3K and SHP-2 signaling were critical for cell transformation in vitro. When various Ink4a/Arf–/– mAsts were separately implanted into the brains of mice, PDGFRα-R627, PDGFRα-F731/42, PDGFRα-F720, and PDGFRα-F988 significantly impaired PDGFRα-promoted tumorigenesis and invasion compared with WT PDGFRα (Figure (Figure6,6, B, C, E, and F, compared with Figure Figure6A).6A). However, mAsts expressing PDGFRα-F572/74 or PDGFRα-F1018 displayed tumorigenicity comparable to WT PDGFRα tumors (Figure (Figure6,6, D and G, compared to 6A), but showed markedly reduced tumor invasion compared with the tumors derived from mAsts expressing WT PDGFRα (Figure (Figure6,6, K and N, compared with 6H). Higher-magnification images of PDGFRα-R627, PDGFRα-F731/42, and PDGFRα-F720 mutants (Figure (Figure6,6, I, J, and L) further illustrated that these mutations significantly negated the enhanced tumorigenicity conferred by PDGFRα activation, leading to the formation of tumors similar in volume and invasiveness to those seen in GFP-expressing mAst tumors (Figure (Figure1,1, D and I). Moreover, PDGFRα-F731/42 and PDGFRα-F720 tumors showed a significantly decreased cell proliferation (Figure (Figure6,6, O and P) and increased apoptosis (Figure (Figure6,6, Q and R), whereas PDGFRα-F572/74, PDGFRα-F988, and PDGFRα-F1018 tumors exhibited moderate or minimal impacts on cell proliferation and survival compared with WT PDGFRα tumors. Conversely, retention of any 1 of the 5 signaling modules (Y731/42, Y572/74, Y720, Y988, and Y1018 PDGFRα mutants) (10) was insufficient to rescue the abolished tumorigenesis in the brain (data not shown). Taken together, these data indicate that ablation of PDGFRα association with SHP-2 or PI3K abrogates tumorigenicity of Ink4a/Arf–/– mAsts expressing PDGFRα, whereas individual association of PI3K, SHP-2, SFK, or PLCγ with the RTK was insufficient to elicit the full spectrum of tumor-promoting effects of PDGFRα.
We further investigated whether inhibition of SHP-2 and PI3K activities by pharmacological approaches suppresses tumorigenicity of PDGFRα/PDGF-A–expressing Ink4a/Arf–/– mAst and LN444 cells. To this end, we exploited several pharmacological inhibitors against PI3K (LY294002), SHP-2 (PHPS-1 and NSC87877) (23), and SFKs (SU6656, and PP2). As shown in Figure Figure7,7, LY294002 inhibited PDGF-A–induced phosphorylation of Akt at 10 μM in mAsts and 5 μM in LN444 glioma cells, whereas Erk1/2 phosphorylation was unaffected by LY294002 treatment in both types of cells (Figure (Figure7,7, A and B). Of note, a modest decrease was observed in p-Erk1/2 levels in mAsts treated with 20 μM LY294002 (Figure (Figure7A).7A). At a concentration of 100 μM (23), both PHPS-1 and NSC87877 significantly inhibited p-Erk1/2, a direct downstream target of SHP-2, in mAsts and LN444 cells (Figure (Figure7,7, A and B). Next, we examined the impact of the pharmacological interventions of these signaling enzymes on in vitro cell transformation and found that 10 μM LY294002, which had no effect on Erk1/2 activation, suppressed soft agar growth of PDGFRα-expressing mAsts stimulated by PDGF-A (Figure (Figure7A)7A) and PDGF-A–expressing LN444 cells (Figure (Figure7B).7B). Similarly, 100 μM of either PHPS-1 or NSC87877 ablated PDGFRα-stimulated, anchorage-independent growth in soft agar but only had a minimal effect on the cell survival and caspase-3 activation of both PDGFRα/PDGF-A–expressing mAsts and LN444 cells in culture (Supplemental Figure 4, A and B). In contrast, 2 SFK inhibitors, SU6656 and PP2 each at 5-μM concentration, only showed a modest impact on tumorigenesis of these cells (Figure (Figure7,7, A and B). Additionally, the MEK inhibitor PD98059 at 10 μM, which suppressed PDGF-A–induced p-Erk1/2 in mAsts (Supplemental Figure 4C), also inhibited soft agar growth of these cells (Figure (Figure5A),5A), validating Erk1/2 as a mediator of PDGFRα–SHP-2 signaling. Taken together, these data demonstrate that the PDGFRα/PDGF-A–enhanced tumorigenicity of Ink4a/Arf-deficient mAsts and human glioma cells requires intact SHP-2 and PI3K enzymatic activities.
We then examined the impact of SHP-2 inhibitors (PHPS-1 and NSC87877) and PDGFRα-F720 on PI3K signaling. As shown in Figure Figure5B5B and Figure Figure8A,8A, PDGFRα-F720 mutant and SHP-2 inhibitors significantly decreased PDGF-A–stimulated p-Akt in PDGFRα-expressing mAsts. A nearly complete knockdown of SHP-2 by siRNAs markedly reduced the stimulated p-Akt levels in these cells, possibly through interruption of the association between PI3K (p85 subunit) and PDGFRα (Figure (Figure8B).8B). However, a previous study showed that PDGFRα-F720 mutation did not result in a decrease in PI3K association with the RTK in mouse fibroblasts (10). Similarly, when SHP-2 was knocked down in NIH3T3 cells, a minimal impact of SHP-2 inhibition on PDGF-A–induced PI3K association and Akt phosphorylation was observed in these fibroblasts (Supplemental Figure 5A), suggesting that the impact of SHP-2 on PI3K association with the RTK and p-Akt in astrocytes and glioma cells was specific. To examine whether the effect of SHP-2 knockdown on p-Akt signaling was specific to PDGFRα signaling, we knocked down SHP-2 by siRNAs in EGFRvIII-expressing Ink4a/Arf–/– mAsts (WT PTEN) and LN444 and U87MG (both PTEN mutant) cells. We observed a reduction or a modest impact of p-AKT level in LN444/EGFRvIII and U87MG/EGFRvIII cells, respectively (Supplemental Figure 5B), suggesting that SHP-2 also regulates PI3K/AKT activation in other RTK signaling. However, Akt phosphorylation was absent in Ink4a/Arf–/– EGFRvIII mAsts, possibly due to the presence of WT Pten in these cells.
We next tested whether signaling downstream to Akt was affected by SHP-2 inhibition. We found that SHP-2 inhibitors (Figure (Figure8A),8A), SHP-2 siRNA knockdown (Figure (Figure8B),8B), or PDGFRα-F731/42 and PDGFRα-F720 mutations (Figure (Figure8C)8C) significantly impaired PDGF-A–stimulated phosphorylation of S6 kinase (p-S6K) downstream to the mammalian target of rapamycin (mTOR) pathway. Thus we further determined the impact of the ablation of SHP-2 and its downstream target the mTOR pathway on cell transformation. Strikingly, stable knockdown of SHP-2 by shRNAs markedly reduced PDGFRα-promoted soft agar growth of mAsts (Figure (Figure8D).8D). Additionally, treatment with mTOR complex 1 (mTORC1) inhibitor rapamycin led to a dose-dependent decrease in soft agar colonies formed by PDGF-A–stimulated Ink4a/Arf–/– mAsts expressing PDGFRα or LN444 cells (Supplemental Figure 6, A and B), compared with their respective DMSO-treated controls. Similarly, rapamycin was able to effectively suppress the cell transformation capacity conferred by EGFRvIII expression in these cells (Supplemental Figure 6C). Previous studies suggested that the limited efficacy of rapamycin in clinical use was due to the capacity of rapamycin to potentiate PI3K/Akt signaling (24). However, we did not observe an increase of p-Akt level in both Ink4a/Arf-deficient mAsts and LN444 cells treated with rapamycin up to 72 hours (Supplemental Figure 6D). These results suggest that PDGFRα-promoted tumorigenesis necessitates an intact SHP-2 activity that regulates the PI3K/AKT/mTOR pathway. Since, in clinical glioblastomas, constitutively active PI3K mutations are mostly observed in specimens with no PDGFRA aberrations (3, 4), we introduced a constitutively active PI3K p110 subunit (p110α-CAAX) into Ink4a/Arf–/– mAsts that either expressed PDGFRα-F720 or an SHP-2 shRNA. In both cell lines, we observed a rescue effect of p110α-CAAX in soft agar colony formation (Figure (Figure8E),8E), suggesting that PI3K/AKT/mTOR acts downstream of SHP-2 in PDGFRα-overexpressing gliomas, and that activating PI3K mutations in clinical gliomas might be able to bypass SHP-2 activation to promote tumorigenesis. Taken together, our results suggest that SHP-2 regulates the PI3K/AKT/mTOR signaling emanating from PDGFRα activation.
To determine whether PDGF-A and PDGFRα are co-expressed and whether there is a link of overexpression of PDGF-A and PDGFRα and activation of the SHP-2 and the PI3K/AKT/mTOR signaling in clinical glioma specimens, we performed IHC staining on a total of 158 paraffin-embedded primary human glioma specimens using anti-PDGFRα, anti–PDGF-A, anti–p–SHP-2 Tyr542, anti–p-AKT Ser473, and anti–p-S6 Ser235/236 antibodies. PDGFRα proteins were detected at medium to high levels in 17 of 87 GBMs (WHO grade IV tumors) and in 9 of 71 grade II and III tumors. Among these PDGFRα-positive gliomas, PDGF-A was often co-expressed in the same population of tumor cells (Figure (Figure9,9, B and C, and Supplemental Table 1). Significantly, phosphorylation of SHP-2 Y542 (p–SHP-2, required for activation of SHP-2) (25), AKT (p-AKT), and ribosomal S6 subunit (p-S6) was also often detected on sister sections of the same tumor in many of the PDGF-A/PDGFRα–positive glioma specimens (Figure (Figure9,9, B–F, and Supplemental Table 1), suggesting a link between activation of PDGF-A/PDGFRα and stimulation of SHP-2, AKT, and mTOR in clinical glioblastoma specimens. To validate these data, we performed IB analyses on a separate cohort of a total of 20 snap-frozen clinical GBM specimens. As shown in Figure Figure9G,9G, PDGFRα was expressed at high levels in 7 of 20 GBM samples, 5 of which expressed PDGF-A proteins, suggesting an autocrine PDGFRα signaling in these tumors. In 4 of these 5 PDGF-A/PDGFRα–positive tumors, p-AKT, p–SHP-2, and p-S6 were also detected. Of note, expression of PDGF-A, p-AKT, p–SHP-2, and p-S6 at various levels in other tumor samples likely reflects the impact of heterogeneous gene alterations such as mutations of PTEN, TP53, and overexpression of EGFR/EGFRvIII and c-MET that affect the expression or activation (phosphorylation) of these proteins in clinical glioblastomas. Taken together, these results establish a link of PDGF-A/PDGFRα expression with activation of SHP-2 and PI3K/AKT/mTOR signaling in clinical glioblastoma samples.
In this study, we report that PDGFRα and/or PDGF-A overexpression is able to drive gliomagenesis of Ink4a/Arf-deficient mAsts and human glioma LN444 and LN443 cells. Re-introduction of p16INK4a but not p19ARF into Ink4a/Arf-null mAsts suppresses PDGFRα-promoted tumor growth. In the absence of PI3K or SHP-2 signaling, PDGFRα fails to enhance tumorigenesis in the brain of mice. Additionally, we establish a link between activation of SHP-2 and the PI3K/AKT/mTOR signaling in PDGFRα-stimulated tumorigenesis in vitro, in mice, and in clinical glioblastoma specimens. Therefore, our data demonstrate that co-alteration of the RTK PDGFRα and tumor suppressor p16INK4a is required for gliomagenesis and that SHP-2 is a critical linker between the PI3K/AKT/mTOR pathway and PDGFRα in the formation of gliomas.
A unique feature of this study is that specific activation of PDGFRα signaling in vivo by PDGF-A, a ligand that binds to PDGFRα but not PDGFRβ (14) as an autocrine loop significantly enhanced the tumorigenesis of Ink4a/Arf-deficient mAsts and human glioma cells in the brain. Early studies of clinical glioma specimens showed that PDGF-A and PDGFRα are overexpressed in tumor cells, while PDGF-B and PDGFRβ are expressed in hyperplastic capillaries, suggesting both autocrine and paracrine loops for PDGF/PDGFR activation in gliomas (15). In neonate and adult mice, expression of PDGF-B induces de novo gliomas from GFAP-positive astrocytes and nestin-expressing glial progenitor cells through activation of PDGFRα and PDGFRβ in the brain of both WT and Ink4a/Arf-deficient animals (26, 27). Moreover, in WT Ink4a/Arf mice, infusion of PDGF-A proteins into the lateral ventricle stimulated tumor-like growth of PDGFRα-positive NSCs in the SVZ in the brain (17). Importantly, data from The Cancer Genome Atlas (TCGA) and other studies revealed that PDGFRα is overexpressed and amplified and often co-expressed with PDGF-A in clinical glioblastoma samples (3, 28, 29). Our results not only functionally validated these studies but also further demonstrated the significance of specific activation of PDGFRα signaling by PDGF-A in cooperation with loss of p16INK4a but not p19ARF in promoting gliomagenesis. We found that when PDGFRα signaling is activated in Ink4a/Arf–/– mAsts or human glioma cells, mice that received these cells developed significantly larger and highly invasive tumors in the brain. In contrast, no enhancement of tumorigenesis was found in mice that received glioma cells with PDGFRα and an intact CDKN2A locus (19). Together, our studies indicate that PDGFRα activation together with Ink4a/Arf loss results in enhanced tumor growth of both mAsts and human glioma cells in the brain.
Tumor suppressor P16INK4A is frequently mutated in clinical glioblastomas (3, 4, 29). In mice, loss of p16INK4a and p19ARF was shown to be indispensable in facilitating tumorigenesis (6). Ink4a/Arf-deficient mice were viable and developed spontaneous tumors at early ages, but without detectable tumors in the brain (6, 7). Further studies showed that Ink4a/Arf loss cooperates with oncogenic K-Ras, EGFRvIII, or PDGF-B expression in promoting gliomagenesis in the brain (7, 8, 16, 26, 27). However, compared with p16INK4a loss, which contributes to tumor initiation from mAsts, p19ARF deficiency was shown to display a more pronounced impact on cell transformation and gliomagenesis (30, 31). Our data corroborate and also differ from these studies. We showed that re-expression of p16INK4a but not p19ARF in PDGFRα-expressing Ink4a/Arf–/– mAsts inhibited tumorigenesis. Inhibition of CDK4/6, the direct target of p16INKa by a specific inhibitor (20), in Ink4a/Arf–/– mAsts and glioma cells attenuated PDGF-A stimulation of soft agar growth, suggesting that CDK4/6/p-RB signaling is required for PDGFRα-induced tumorigenesis. On the contrary, although p53 was functional in Ink4a/Arf–/– PDGFRα-expressing mAsts, p19ARF was unable to suppress soft agar growth of these cells. Since p19ARF de-represses p53 signaling while PI3K/Akt activates p53 E3 ubiquitin ligase Mdm2 (32), it is plausible that the robust PI3K/Akt activation in PDGFRα-overexpressing cells triggers Mdm2-mediated p53 degradation and thereby renders tumorigenic mAsts resistant to p19ARF inhibition (21). It is also likely that loss of p19ARF is required for survival of glioma cells under certain situations, such as treatment of DNA damage–inducing agent CDDP (Supplemental Figure 3). At least during the initiation or maintenance of cell transformation, p19ARF loss appears to be dispensable (30, 31), since knockdown of INK4A alone in WT INK4A/ARF LN319 glioma cells restored PDGFRα stimulation of anchorage-independent growth in soft agar. Taken together, these data suggest that loss of p16INK4a plays a predominant role in PDGFRα-promoted gliomagenesis.
The third important aspect in this study is that we functionally assigned signal modules of PDGFRα in promoting gliomagenesis and tumor invasion in the brain of mice. Previous studies by uncoupling individual signaling pathways from PDGFRα using a series of F-to-Y mutants revealed unequal contributions of each signaling pathway emanating from PDGFRα activation (11, 12, 14). Our in vivo and biochemical data corroborate these reports. When compared with WT PDGFRα overexpression, loss of intrinsic tyrosine kinase activity (R627) of the RTK or binding capacity to PI3K (F731/42) or SHP-2 (F720) abrogated PDGFRα-promoted gliomagenesis, thus signifying the central roles of PI3K and SHP-2 signaling in PDGFRα function. However, while disruption of PDGFRα association with SFKs (F572/74) or PLCγ (F1018) only had a moderate impact on tumor formation in the brain, significant inhibition of tumor cell infiltration in the brain was seen in these tumors, as compared with the WT PDGFRα tumors, thus validating the role of SFKs and PLCγ in mediating PDGFRα-stimulated cell invasion (10). Moreover, a separate set of experiments with individual "add-back panel" mutants in which 1 of 5 signal modules was individually retained as single or double Y residues (Y731/42, Y572/74, Y720, Y988, and Y1018) could not fully restore PDGFRα-promoted tumorigenesis in the brain (data not shown), consistent with the previous findings using an identical set of PDGFRα add-back panel mutants (10). It is plausible that, similar to this previous in vitro study (10), in which retention of a combination of 2 or 3 add-back signaling modules with comparable levels of total RTK protein in WT PDGFRα–expressing cells was able to restore PDGFRα-mediated biological responses, co-activation of PI3K (Y731/42)– and SHP-2 (Y720)–mediated signaling may be required for the full spectrum of WT PDGFRα–promoted gliomagenesis in the brain.
The last (and what we believe is also the most novel) finding in this study is the emergence of SHP-2 as an essential mediator in PDGFRα-promoted gliomagenesis. SHP-2 (encoded by PTPN11 gene) is a protein tyrosine phosphatase (PTP) identified as a bona fide proto-oncogene that activates Ras/MAPK signaling through a yet-to-be-defined mechanism (33). Additionally, the role of SHP-2 in mediating PDGFRα signaling has not been clear (9, 11, 14). In human cancers including GBMs, mutations of SHP-2 or its binding partners have been reported, leading to sustained Ras/MAPK signaling (34, 35). Recent genomic analysis of TCGA data has designated PTPN11 as one of the 6 "linker" genes, which are statistically enriched for connections to various GBM altered genes, thus suggesting a critical role for SHP-2/PTPN11 in modulation of downstream biological signaling in gliomagenesis (36). Our data not only establish the critical role of SHP-2 in mediating PDGFRα activation for glioma formation but also functionally validate this hypothesis. We showed that inhibition of SHP-2 function by removal of its binding module in PDGFRα (F720 mutant), gene knockdown or pharmacological inhibitors significantly impaired PDGFRα stimulation of tumorigenesis in vivo and in vitro and its downstream signaling effectors, Erk1/2 and PI3K/Akt/mTOR in Ink4a/Arf-deficient mAsts and glioma cells. Significantly, re-expression of a constitutively active p110, the catalytic subunit of PI3K, rescued the inhibition of SHP-2 in Ink4a/Arf-deficient mAsts. In EGFRvIII-expressing U87MG glioma cells, SHP-2 regulates activities of ERK/2 and CDC2 that modulate cell cycle progression but has a minimal effect on AKT activation (23). On the contrary, we found that knockdown of SHP-2 not only attenuated PDGFRα stimulation of Erk1/2 activity but also impaired PI3K/Akt/mTOR activity in Ink4a/Arf-deficient mAsts and human glioma cells. The impact of inhibition of SHP-2 on PI3K/Akt signaling appears to be specific in astrocytic tumors, since we did not observe inhibited PDGF-A stimulation of p-Akt or association of PI3K with PDGFRα in SHP-2–knockdown NIH3T3 fibroblasts. On the other hand, the MEK inhibitor PD98059 also reduced the tumorigenicity of Ink4a/Arf-deficient mAsts, indicating that SHP-2–mediated PDGFRα signaling requires not only PI3K/Akt but also Erk1/2 activation in gliomagenesis. Additionally, SHP-2 was found to either positively or negatively regulate PI3K/AKT activity (34). However, in our model systems, SHP-2 is required for full activation of PI3K/Akt/mTOR, and inhibition of mTOR by rapamycin markedly suppressed PDGFRα-promoted tumorigenesis. These data are significant since a recent proteomic study revealed that mTOR signaling is predominantly activated in "PDGFRA co-cluster" glioblastomas (37), thus corroborating our observations. However, since phosphorylation of Akt Ser473 occurs both upstream and downstream of mTORC2 signaling, our data do not rule out the role of mTORC2 in PDGFRα-activated signaling. Lastly, our data also demonstrated the importance of activation of PI3K/mTOR signaling by SHP-2 in PDGFRα- or EGFRvIII-promoted tumorigenesis. Taken together, our findings suggest SHP-2 as a critical modulator that regulates PDGFRα-mediated PI3K/AKT/mTOR activities in the development of malignant glioblastomas.
In summary, this study provides molecular insights into the mechanisms by which PDGFRA amplification together with loss of INK4A/ARF promotes gliomagenesis in the brain. Our data identified SHP-2, as well as PI3K, as a pivotal mediator of PDGFRα signaling in glioma formation. These results have direct clinical relevance, since we not only establish a model system to demonstrate the co-operative role of PDGFRA overexpression and INK4A/ARF loss in clinical glioblastomas, but also provide functional evidence to validate genomic analyses demonstrating that SHP-2/PTPN11 is an essential "linker" among glioma altered genes (36). Secondly, activated mTOR signaling is predominantly found within the subclass of glioblastomas with abnormal PDGFRα signaling (37). Finally, constitutively active PI3K mutations (PIK3CA and PIK3R1) occur mostly in clinical glioblastomas without PDGFRA aberrations (4). Consequently, our results strongly suggest SHP-2/PTPN11 as a potential target for treatments of glioblastomas with PDGFRA overexpression.
Primary Ink4a/Arf–/– mAsts were derived and propagated as previously described (7). Human glioma cell lines LN444, LN443, LN-Z308, and LN319 were obtained from ATCC or from our own collection (38). Unless otherwise mentioned, all glioma cell lines and primary mAsts were routinely maintained in 5% CO2 at 37°C, in DMEM (Invitrogen) containing 10% FBS (Hyclone), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen).
Studies using human tissues were reviewed and approved by the Institutional Review Board involving Human Subjects of the University of Pittsburgh. The specimens were de-identified human tissues, and thus no informed consent was required. A total of 158 paraffin-embedded thin sections of primary human glioma specimens were used, including 87 WHO grade IV GBMs, 34 grade III anaplastic astrocytomas, anaplastic oligodendrogliomas, or anaplastic oligodendroastrocytomas, and 37 grade II oligodendrogliomas and diffuse astrocytomas. Thin sections of human glioma specimens and mouse brains with various tumors were analyzed by IHC using indicated (e.g., Figure Figure88 legend and Results) antibodies or a TUNEL staining kit as previously described (38, 39). Briefly, the 5-μm human tissue sections were deparaffinized in xylene, followed by rehydration in graded ethanol. After washing with TBS, the antigen was retrieved by boiling the sections in a citrate buffer (pH 6.0) twice for 5 minutes. For mouse brain tissues with various gliomas, 5-μm frozen sections were fixed in pre-chilled acetone at –20°C for 5 minutes, rinsed with PBS, and blocked by AquaBlock (East Coast Biologics Inc.) for 1 hour at room temperature. Afterward, various tissue sections were then incubated with a primary antibody overnight at 4°C and blocked by Peroxidase Blocking Reagent (DAKO) for 10 minutes, followed by incubation with a biotinylated secondary antibody for 30 minutes at room temperature. After washing in PBS, stained tissue sections were visualized by diaminobenzidine chromophore and H2O2, followed by hematoxylin counterstaining. Sections were then dehydrated by graded ethanol and mounted with Permount Solution (Fisher).
Colony formation assay in soft agar was performed as previously described (40). Briefly, approximately 5,000 cells were seeded in a 0.5% Noble Agar top layer with a bottom layer of 0.8% Noble Agar in each of the triplicate wells of a 24-well plate. Growth factor–reduced Matrigel (1 mg/ml) was added into the top layer with or without inhibitors as indicated in figure legends (e.g., Figure Figure77 legend) and described in Results. PDGF-A–expressing cells in 10% of total cells were included as a source of PDGF-A. DMEM containing 10% FBS was added 3 days after plating and changed every 3 days thereafter. Colonies were scored after 2–3 weeks using Olympus SZX12 stereomicroscope, and data were analyzed using GraphPad Software.
Experiments of mouse glioma xenografts, IP, IB, tissue imaging and analyses, and siRNA knockdown were performed as previously described (39). For details, see Supplemental Experimental Procedures.
One-way ANOVA or an unpaired, 2-tailed Student's t test followed by Newman-Keuls post-test was performed using GraphPad Prism software. A P value of 0.05 or less was considered statistically significant.
We would like to thank Carl-Henrik Heldin at Uppsala University for PDGFRα and PDGF-A cDNAs, Russell O. Pieper at University of California San Francisco for pMXI-gfp retroviral vector, Lynda Chin at Dana-Farber Cancer Institute for the cDNA of p16INK4a, Yi Zheng at Cincinnati Children's Hospital Medical Center for cDNA of p19ARF, Tao Cheng at University of Pittsburgh for pMSCV retroviral vectors, and N. Balass for proofreading the manuscript. This work was supported by grants from the NIH (CA102011 and CA130966 to S.-Y. Cheng; EY012509 to A. Kazlauskas; CA87375 to K. Symes), American Cancer Society (RSG CSM-107144), and Pennsylvania Department of Health and Innovative Research Scholar Awards of the Hillman Foundation (to S.-Y. Cheng and B. Hu).
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2011;121(3):905–917. doi:10.1172/JCI43690.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
Tomica (18 items)
Welcome back! Whilst you were away we added the functionality to view auctions and bid in pounds.
Tomica: Information on the Catawiki site about Mitsubishi Fuso 'Coca-Cola', 1988.
At its heart, Catawiki is an online catalogue with images of, and information on, collectibles. Our catalogue is compiled by users in a similar way to Wikipedia.

subset: commoncrawl


text:
J.Lo is striking while the iron is hot.
The star, who wept and said, "I can't do this anymore!" at the end of last night's American Idol show (7:50 mark in the video) after she had to kick off contestant Chris Medina, will unveil her new music video for single On The Floor during American Idol on March 3. On the Floor marks J.Lo's first new studio release on the Island Def Jam Music Group.
Meanwhile, in other Idol news this morning: Medina will perform on The Tonight Show with Jay Leno on Friday night. And Casey Abrams has been hospitalized with stomach pains.
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Demi Lovato to Ben Stiller: "Buy My New Album!" - Watch Here!
Hitting the streets of the Big Apple to try and sell some records, Demi Lovato tried to make a famous face a Lovatic this week.
While being driven around NYC, the 20-year-old singer spotted Ben Stiller out and about and yelled, "Ben Stiller, buy my new album on iTunes! It's out now, it'll give you a heart attack."
Turning back to her friends in the car, Demi smiled and said "F**k yeah!" before erupting in laughter with her friends. Check out all the fun in the video below.
On Sunday, the former Disney darling thanked another famous fan, tweeting a pic of Nick Jonas posing with a copy of her album in a store. Appreciating the shout-out, Ms. Lovato commented, "Look at this guy... Said he bought every copy they had..!!! Love you @nickjonas, you are the best!!!!"
Photo Credit: Getty Images


text:
Everyone is passionate about something—everyone geeks something. Your public library supports it all. Support your library.
Historically, 'access for all' is what drove the establishment of most public libraries. This mission still rings true today. For many Americans, their local library is an important source for free public access to computers and the Internet in their communities—and for some, it is their only access.
Last year, librarians helped millions of people find out more about what they geek, discover new interests and search for jobs online. Librarians are passionate about your community, they are passionate about what you geek and they are passionate about you.
In addition to traditional library resources, such as books and children's programs, your library offers innovative geeking opportunities for teens and adults. Genealogy research, nonprofit support, early childhood literacy programs and more.
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- About WKU T&D
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It's Time for Something New
Have you been planning a piece of performance art? Dreaming up a solo show? Drafting a ten-minute play? Wishing you could collaborate with a composer on a new movement piece? Imagining actors and/or dancers performing live onstage with a film you've made? Pondering a devised show? Well, here's your chance to make those dreams a reality!
The Department of Theatre and Dance is putting on a New Works Festival April 10 – 15 in the Lab Theatre in Gordon Wilson Hall, and it will be what you make it: this is a chance for student-driven, student-created projects to take center stage!
- No previously produced work.
- You can only be part of two proposals.
- Proposals must come from a WKU student.
- Respect copyrights.
- Preference will be given to projects between 10 and 20 minutes in length.
Applications are due via email by 5pm on Tuesday, 12/3. Questions? Contact Dr. Michelle Dvoskin.
Download the New Works Festival Application Form
Download the 10 Minute Play Festival Application
Check out a Sample 10 Minute Play
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Community, Translation, and Wapuu: How Japan is Shaping WordPress History
Japanese WordPress users were some of the earliest to see the project's potential and help bring the software to the non-English speaking world. At the end of 2003, just six months after Matt Mullenweg and Mike Little decided to fork b2, a Japanese version of WordPress was available. The version (more…)


text:
Volume Pot for RH84
2 Attachment(s)
Just finishing up my RH84 build and wanted to see if anyone had used a 100K pot for a volume control.
The schematic shows a 1 Meg resistor on the input. If I put a 100K pot across that, the resistance will drop. Is that something to be concerned with?
BTW, this project started life as a Dumont console. I reused the transformers and output tubes. You can see the before and after photos.
100k is perfect.
Sweet build ......... :cool:
Ya, how do you like it?
Still not finished -- Hopefully tomorrow. I just hope there aren't any problems and it sounds great.
Soldered up the 100k pot. Thanks for the confirmation Bas.
Finished the amp last night. It sounds great. Plenty of power and a nice clean sound. Spent the day listening.
There was one hiccup. When I first powered it up, all the votages looked normal. When I added the 12AT7 and turned it on, there was some nice blue sparks in the tube and and a curl of smoke. I found the grid and cathode resistors fried. I also noticed that there was a wire from the output transformer that looked like it might be touching the input end of the grid resistor for one channel. In the original circuit, there was an extra winding on the primary side of the output transformer that was used as a filter choke in the power supply. That wire probably had about 300V on it. It was clipped, but I guess that there was enough metal to make contact with the resistor lead. After I cleaned it up and replaced the resistors, it worked perfectly. Oh, I had to replace the 12AT7 too. Guitar Center to the rescue.
I'm very pleased. The amp sounds great and looks good too.
Right on, it is a nice project, glad everything worked out for you.
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The better tool for cutting/splicing romex?
This? http://www.amazon.com/Ridgid-21438-R...5552764&sr=8-7
This? http://www.amazon.com/45-025-IDEAL-L...5552817&sr=8-1
It seems like it can do everything the more expensive tool can. Has anyone used both of these that can help?
I've got a pair like the top picture--They are made by Kline----I like them and have had a few others run out to buy them after they borrowed mine.
I am not in a Romex area so I don't have much use for them--But they do work well.---Mike---
That said, I've never used the "Lil Ripper Stripper" but don't believe there is any way that a plastic $5 tool is going to do the job you want it to. In fact, I doubt seriously that it will work at all.
Actually, mine aren't that "fancy", but if the second one is what I believe it is, it's not an either/or thing. I believe that the second one has a blade of sort inside it, which is for the purpose slicing the outer sheathing on NM or "Romex" cable, so that you can access the individual wires. Afer you have sliced the sheathing with that tool, and cut it out of the way, you would use the first tool, or something similar, to strip, cut, and bend the individual wires.
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Bruise It or Lose It
Sun 8/24
This weekend is World Wrestling Entertainment's latest installment of the SummerSlam pay-per-view event. One of WWE's biggest annual mega-bouts, it features wrestlers from both RAW and SmackDown!. But for around the price of a pay-per-view purchase, you can roll on down to America West Arena and experience it firsthand. This year's SummerSlam will have no shortage of drama -- nothing less than you'd expect from Vince McMahon and his cronies. To wit: After Eric Bischoff defeated Shane McMahon on RAW several weeks ago, Bischoff thought he'd seen the last of McMahon but then inadvertently signed on to battle him again at SummerSlam. Also, in the biggest grudge match of the event, Rob Van Dam (affectionately known as the Big Red Machine) will take on former tag-team partner Kane, who was recently unmasked after years of making like an enigma. SummerSlam kicks off at 4:45 p.m. Sunday, August 24. For ticket info, call 480-784-4444. - Brendan Joel Kelley
Howl Play
Answer the call of the wild during the Coyote Walk
Fri 8/22
Walk by the light of a silvery moon at the Riparian Institute's Coyote Walk, 8 p.m. Friday, August 22. A crescent moon makes for starry skies and encounters with not only coyotes, but also bats, scorpions, and the odd insomniac heron fishing for a snack. It's peaceful out there (unless you're prey), and institute director Scott Anderson knows where the wild things are.
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So what do coyotes do all night? Same as us -- scare up dinner, hang with the boys and howl a little on payday. The cost for the walk is $3 per person, reservations recommended. Meet behind the Southeast Regional Library at the corner of Greenfield and Guadalupe roads in Gilbert. For more information, call 480-503-6744 or 602-696-1195. - Kim Toms
Hit Makers
Summerfest makes a racquet
Sat 8/23
Back in the early '80s, the calling cards of speed, skill and nonstop action hearkened the beginning of racquetball's glory days. Times and tastes change, but the sport has always maintained a loyal following among those who prefer their racquet sports with a little more attitude and all the unpredictability four walls, a ceiling and a little blue ball can provide. Make your play for racquetball supremacy at the Summerfest Racquetball Tournament at 11 a.m. Saturday, August 23, at Sports Club at City Square, 4000 North Central, Suite 110. A continuous play "shuffle format" separates the contenders from the pretenders, with awards for the top three finishers and the culinary gift of Honey Bear's Bar-B-Q to be shared among all participants. The entrance fee is $5 for members and $10 for nonmembers. For details call 602-285-2929 or visit www.sportsclubcitysquare.com.- Craig Wallach
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text:
Use Pet Wrap Protect as a way to secure bandages and protect injured areas.
1. Use a clean damp cloth to carefully clean, then bandage the wound.
2. Starting away from the heart, wrap around the wound, overlapping the previous layer by half. Tear or cut to size.
3. If your pet is in extreme pain or is bleeding, ask your veterinarian for advice.
"Nothing holds better then pet wrap. I use it on all my patients."
Dr. Simon George. Deerfield Veterinarian Clinic


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Give your garden a classic Indian Limestone patio. This natural stone slab with its soft Brown hue exudes refined calm. The product is hand-cut which only adds to the earthy natural finish. Use this pack to create a striking patio effect.
Add another dimension to your patio project by adding some handsome Sandstone Brown Cobble.
Hand-picked to complement the item you're viewing.
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Reviewing: Kota Brown Hand-Cut Indian Limestone Patio Pack A
by Timmy Sheehy on 13 Feb 2014
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Fax: 01 4589999
Tile Office Tel: 01 4019052
Email: firstname.lastname@example.com
Tile Office Mon -> Fri onlyRegistered Company No: 305205 © 2017 Deeward Ltd T/A Stone Merchant


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|Attractions/Entertainment, Parks/Recreation
Broadway & Santa Monica Blvd & 3rd St
Santa Monica, CA 90401, US
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Our Wedding
Randi and Michael's Wedding
"Shopping, dining, movie theatres, street performers... one of the few places in LA you can actually walk around."
Rachel and Tom's Wedding
"Drawing locals and world travelers alike, Downtown Santa Monica is a popular Southern California coastal destination, anchored by the world-famous Third Street Promenade. Known for a twice-weekly farmers market, fabulous dining and terrific shopping, Downtown also has lots of entertainment choices from movies to live theater to street performers. All this just minutes from beaches and the Pacific Ocean."
Jessica and Johnny's Wedding
"Check out the downtown Santa Monica! There are tons to do! But come to our reception on time with empty stomach"
Newbury Park Wedding In June
"This is a fun outdoor shopping area that usually has street performers on the weekend. It's within walking distance of the beach. It's very "SoCal""
Fountain Valley Wedding In July
Santa Monica Wedding In February
Long Beach Wedding In August
Third Street Promenade
Broadway & Santa Monica Blvd & 3rd St
Santa Monica, CA 90401, US


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Your Dog, Your Life
The Scoop
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Gear and Glam
Wildebeest creates the perfect present for new dog parents.
Dog Breeds
Foods & Treats
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Photo Comments
The Rules:
Each player starts with 7 random facts about themselves. Dogs who are tagged need to post in their Diary, "The Rules" and their "7 pawsome Facts."
Then choose 7 dogs to tag & list their names. Don't forget to bark them a pmail that they have been tagged and to read your Diary. Or send them a fun Rosette announcing they've been tagged.
Lucian Facts
7. I will eat anything on the ground
6. Going "bye bye" is awfully cool
5. I've been neutered for 7 years but still like to try to hump girls
4. I am afraid of thunder
3. Dancing with the Stars is one of my fav programs to watch w/Mom
2. I do not like to lay on the ground and prefer the couch or a chair
1. I wish all shelter animals could find loving homes, I was a lucky one
Pups Lucian tagged:
Marley (Mastiff)
Nixon (Doberman)
Acuzio (Fila)
Hollie (Husky/Shepherd X)
Archibald Bartholomew (Mastiff)
Lucian (Husky/Retriever X)
Cherokee Rain (Doberman)
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Who are the movers and the shakers of Tokyo's Social Computing scene?
Posted by Site Administrator on October 23, 2008
I had a very enjoyable evening yesterday at the Bloggers' Dinner in Tokyo. It was great to speak with the group of about 30 Japanese and non-Japanese bloggers who had so many interesting perspectives to share. We met at Fujimama's near Omotesando - an ideal venue for this kind of open networking evening.
Jeremiah Owyang introduced the evening with his observations that Japanese consumers are ahead of Japanese companies in their adoption of Social media - Whereas consumers are eagerly leveraging new media to express themselves and interact with each other, Japan's corporate world seems rather hesitant.
Forrester's Technographics data shows that Japanese consumers are eagerly participating in social media as creators, critics, collectors, joiners and spectators.... so why aren't Japanese firms putting more effort into engaging with customers via social media? Here are three thoughts or recommendations that came to my mind. If you disagree, or if you have better recommendations, please post a comment.
With regard to Social Computing - what aren't Japanese companies doing that they should be doing?
- Let go of the "command and control" Public Relations mentality
- The PR and corporate communications departments in Japanese companies want to have complete control over the message
- Japanese companies see Social Media as frightening, threatening places where irresponsible writers make libellous comments with impunity
- Even when large Japanese companies make an outreach to bloggers - as Nissan does by inviting automotive bloggers to vehicle launch events - the companies still want to have a one way flow of information (official communications go out) rather than allowing a dialogue to arise freely.
- Focus on PEOPLE not on technology
- The last thing firms should do in their social media strategy is choose a technology
- Companies first need to understand who their customers are and how they use social media in their lives. Do your target customers want to interact with each other in social networks? or share ideas through some kind of Wiki? When you can see what they're doing, it becomes possible to understand where you need to be to reach them.
- If your target audience is heavily into social networking, then the place to meet with them and interact with them may be in Mixi or Gree. For younger and more "game oriented" audience then the place to find them may be Mobage Town.
- Empower young Japanese employees as innovators
- Kenichi Ohmae has railed against the seniority-based hierarchies in Japanese firms for many years. Simply put, the young employees are the ones who "get" social media. They are the ones who should be empowered to start up new ventures with innovative ideas.
- Japanese corporate culture is such that one person may "veto" the good ideas of the rest of a team. As a result, new business models get stifled and progress takes a long time.
Those are the ideas that came to my mind during my discussions yesterday evening. What do you think?
If you're interested in seeing who the "movers and shakers" are in Tokyo (at least, the ones who came to our gathering), please check out the blogs in the list below. They represent some of the brightest commentators across a variety of fields - culture, business, food, fashion, technology, politics - just about everything:
Bloggers who attended the Oct 22 Bloggers' Dinner
If you attended the event but not on this list, please let me know and I'll add you in here
I will admit, there's a serious gender bias in the list - It's mostly men.
Finally - We made an important announcement last night:
GROUNDSWELL -- The best selling business book by Josh Bernoff (Forrester analyst) and Charlene Li (former Forrester analyst) will be published in Japanese by Shoeisha. I'm really delighted that this book will help us to introduce more Japanese executives to Forrester's ideas and data on Social Computing.
re: Who are the movers and the shakers of Tokyo's Social Comput
Comment from Desozza (not verified) on Mon, 11/24/2008 - 12:20
well… i visit your website first time and found this site very usefull and intresting !well… you guys doing nice work and i just want to say that keep rocking and keep it up !!!!RegardsDesozza
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text: Morey's Piers is Going to the Dogs at Raging Waters Water Park once again! On September 10, 2017 your pooch can paddle in the pools of Raging Waters Water Park at Morey's Piers "Gone to the Dogs"! Man's best friend can enjoy an evening swim from 5:30PM – 7:00PM (check in starts at 5:00pm) at Raging Waters Water Park, located at the rear of Morey's Mariner's Pier at Schellenger Ave. and the Boardwalk in Wildwood, NJ.


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Semiformal Evening Truffles RecipeRead Reviews Be the first to add a review
- 4 cups (24 ounces) semisweet chocolate chips
- 1 package (8 ounces) cream cheese, softened
- 3 tablespoons instant coffee granules
- 2 tablespoons water
- 1 cup sweetened shredded coconut, finely chopped
- 1/2 cup chocolate sprinkles
- In a large microwave-safe bowl, melt chocolate chips. Add the cream cheese, coffee granules and water; beat until smooth. Cover and refrigerate for 45-60 minutes or until thickened, stirring every 15 minutes.
- Shape into 3/4-in. balls; roll in coconut or sprinkles. Store in an airtight container in the refrigerator. Yield: about 3 dozen.
Originally published as Semiformal Evening Truffles in Taste of Home April/May 2008
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Join Date: Jul 2001
Posts: 2,419
Saturday, April 16, 2011 at 11:37PM
^^^ so sad, sinistral.
Dumb rant but yesterday I took my daughter to an Easter egg hunt put on by my husband's work. Despite the fact that we had to RSVP, they didn't have nearly enough eggs for all the little ones that were there. My daughter and the other 1-3 year olds each got like two eggs a piece. Maybe because they didn't understand they had to be fast enough picking them up to beat out the adult lady who picked up about 9 or 10 for her own little boy who was too young to do it himself...OMG, some people just are clueless.


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Bob's All-NBA First Team...No Room For Carmelo Anthony
Do you think New York Knicks star Carmelo Anthony is a worthy All-NBA First-Team selection this season?
I give Anthony credit for playing the best all-around basketball of his NBA career, but I can't give him one of the two All-NBA forward spots over LeBron James (Miami) or Kevin Durant (Oklahoma City).
This is how my All-NBA ballot would look:
FIRST TEAM – F LeBron James (Miami)…F Kevin Durant (Oklahoma City)...C Marc Gasol (Memphis)...G Chris Paul (Los Angeles Clippers)…G Kobe Bryant (Los Angeles Lakers)
SECOND TEAM – F Carmelo Anthony (New York Knicks)...F Tim Duncan (San Antonio)...C Brook Lopez (Brooklyn)...G Russell Westbrook…G James Harden (Houston).
THIRD TEAM – F Blake Griffin (Los Angeles Clippers)...F David Lee (Golden State)...C Dwight Howard (Los Angeles Lakers)…G Tony Parker…G Dwyane Wade.
High honorable mention – Stephen Curry (Golden State)...Paul George (Indiana)...Paul Pierce (Boston)...LaMarcus Aldridge (Portland)...Chris Bosh (Miami).
I'd take Dwight Howard over any other center in terms of talent but he's had a poor season by his lofty standards and hasn't been as consistent as Marc Gasol or Brook Lopez)....Parker was worthy of First-Team consideration before injuries took a toll...Harden is a scoring machine. I won't second-guess anyone who prefers him over Kobe for First Team...Griffin's dunks are awesome, but his overall game needs work. I'm surprised he hasn't shown more progress.
--Will Kevin Durant win his fourth consecutive NBA scoring title or will Carmelo Anthony win his first?
They're locked in the tightest race for the individual scoring crown since David Thompson (Denver) and George Gervin (San Antonio) in the 1977-78 season.
Entering the final day of the season, Gervin was averaging 26.8 points per game and Thompson was averaging 26.6.
Denver played in the afternoon against Detroit and his teammates did all they could to help him win the title. He had 32 points in the first quarter (a new NBA record for one quarter) and 53 points at halftime. He finished with 73 points (28x38 from the field and 17x20 in free throws) in 43 minutes.
San Antonio played a night game at the New Orleans Jazz. The Spurs knew Gervin would win the scoring title if he scored 58 points. No problem. Gervin scored 20 points in the first quarter and 33 points in the second quarter (breaking Thompson's record set several hours before). He finished the game with 63 points (23x49 from the field and 17x20 from the line) in 33 minutes to edge Thompson for the scoring crown (27.22 to 27.15).
There will no such shenanigans between Anthony and Durant. Their teams play their final games at the same time.
Anthony and Durant both played Thursday night. Anthony scored 36 points in a 118-111 Knicks loss in Chicago. Durant had 31 points in a 116-97 home win over Golden State (not surprisingly, teammate Russell Westbrook took more shots and made fewer: Durant 10x16; Westbrook 7x17).
The remaining schedules:
Anthony (Knicks; 4 games) – at Cleveland Friday...host Indiana Sunday...at Charlotte Monday...host Atlanta Wednesday, 8 p.m.
Durant (Thunder; 3 games) – at Portland Friday...Host Sacramento Monday...host Milwaukee Wednesday, 8 p.m.
--Rochester Americans goalie David Leggio did not make the AHL official first- or second All-Star Game. I have lost all faith in the voters. Did they do their homework?
Considering the turnover on Rochester's defense – the veteran stalwarts all are gone (Sabres GM Dacry Regier didn't do much to fill the void and it figures to show in the playoffs) – it is a hockey miracle that the team's record is 39-27-4. I can't imagine a more valuable player in the AHL this season. Leggio leads AHL goalies by wide margins in wins (36), games (59), shots faced, saves and minutes. He ranks 7th in save percentage (.923) and is 8-1 in shootouts.
--Selecting quarterback Ryan Nassib in the first round of the NHL Draft April 25 would be one of the most controversial picks in the history of the Buffalo Bills. One draft talent analyst recently compared Nassib to a young Drew Brees. If he's close to being right, Nassib would be a bargain.
--Probable Kentucky Derby (May 4) favorite Verrazano will try to become the first Derby winner that did not have a two-year-old campaign since Apollo – in 1882. Of course, he doesn't know that.
--The movie 42 – the mostly accurate portrayal of Jackie Robinson breaking baseball's color barrier, opened across the country on Friday. Here are a few Robinson factoids:
He played briefly for the Negro League Kansas City Monarchs in 1945. He hit .414 (24x58).
He was International League MVP in 1946 as second baseman for the Montreal Royals: 124 games; 113 runs; .349 batting average (155x444); 25 doubles; 8 triples; 3 HRs; 66 RBI; 40x55 stolen bases...The Royals played three series in Rochester. Robinson missed the first series because of injury. According to newspaper clips, there were no incidents on or off the field in the subsequent two road series he played against the Red Wings.
He was the National League Rookie of the Year (and fifth in MVP balloting) as first baseman for the Brooklyn Dodgers: .297 batting average (175x590); 125 runs; 31 doubles; 5 triples; 12 HRs; 48 RBI; 27 stolen bases; 74 walks; 36 strikeouts; NL-most 28 sacrifice hits.
His final season was 1956: 117 games; .275 batting average (98x357); 15 doubles; 2 triples; 10 HRs; 43 RBI; 12x17 stolen. The Dodgers traded him to the New York Giants after the season and he retired rather than play for his long-time rivals.
His Major League Baseball career line: 10 seasons; .311 batting average (1,518x4,877); 947 runs; 273 doubles; 54 triples; 137 HRs; 734 RBI; 197 stolen bases.
His numbers are impressive, especially considering the fact that he wasn't allowed to play in Major League Baseball until he was 28 years old.
His top salary was $39,750 in 1952. He was paid $5,000 in his 1947 rookie season and $21,000 in his 1949 league MVP season.
--A few things I'd do to improve college basketball:
Reduce the shot clock to 30 seconds to speed up the action...The shot clock began at 45 seconds in 1985 and was trimmed to 35 seconds in 1993. It wasn't enough.
Junk the NBA rule requiring the very best high school players to play one year in college...Let the cream-of-the-crop dozen-or-so top high school players join the NBA work force immediately...That would be better than having kids play one year in college taking courses such as NBA 101.
Require the other high school players to spend two seasons in college or in foreign countries.
Increase the foul limit from five to six – perhaps with an extra foul shot for opponents after the fifth foul. It would take some pressure off the referee and make it more likely to have the best players on the court when the game ends.
Automatic technical fouls on coaches who are on the court during play on the floor (for example, Rick Pitino).
Bring back the game for third place prior to the NCAA championship game.
--RIP Buffalo Sabres. After an encouraging 2-0 start, it was mostly all downhill. The season will be remembered mostly as the one coach Lindy Ruff got fired – and GM Darcy Regier stayed (at least for now)


text:
Roster of the Food Advisory Committee
Applying for Membership on FDA Advisory Committees
As part of the Food and Drug Administration's (FDA's) ongoing efforts to recruit qualified experts with minimal conflicts of interest who are interested in serving on FDA advisory committees, FDA is requesting nominations for members to serve on its advisory committees.
Current Number of Vacancies: 0
Note, one or more vacancies may be in the nomination process or a final appointment may have been made.
Designated Federal Officer
Karen L. Strambler, M.B.A., M.H.A.
HFS 024-Rm 1C-008
5001 Campus Drive
College Park, MD 20740
Phone: 240-402-2589
Fax: 301-436-2637
E-mail: email@example.com
Kevin L. Armbrust, Ph.D.
Expertise: Analytical Chemistry
Term: 1/10/13 - 6/30/2017
Claiborne Chair and Professor
Department of Environmental Sciences
School of the Coast and Environment
Louisiana State University
1259 Energy, Coast and Environment Bldg.
Baton Rouge, LA. 70803
Leon H. Bruner, Ph.D., D.V.M.**
Expertise: Nutrition and Food Safety
Term: 7/1/2015 - 6/30/19
Senior Vice President and Chief Science Officer
The Grocery Manufacturers Association
1350 I Street NW
Washington, D.C. 20005
Alejandro Castillo, Ph.D.
Expertise: Food Microbiology, Food Science/Technology and Food Safety
Term: 7/28/15 - 6/30/19
Assistant Professor
Texas A&M University
College Station, TX 77843
Sharon M. Donovan Ph.D., R.D.
Expertise: Nutrition and Pediatric Development
Term: 8/26/15 - 6/30/19
Professor and Melissa M. Noel Endowed Chair, University of Illinois at Urbana-Champaign
905 South Goodwin Avenue
Urbana, IL 61801
Richard A. Durst, Ph.D.
Expertise: Food Science, Food
Technology & Chemistry
Term: 8/26/13 - 6/30/17
Professor Emeritus of Chemistry
Department of Food Science & Technology
Cornell University
425 Riley-Robb Hall
Ithaca, NY 14853
Jeffrey M. Farber, Ph.D.
Expertise: Food Microbiology and Food Safety
Term: 10/26/15 - 6/30/18
University of Guelph
Department of Food Science
Guelph, Ontario N1G2N1
Susan V. Grooters, M.P.H.*
Expertise: Epidemiology, Food Science, and Food Safety
Term: 7/1/2015 - 6/30/19
Policy Analyst
Keep Antibiotics Working (KAW)
3525 W. Peterson Avenue
Chicago, IL 60659
A. Wallace Hayes, Ph.D.
Expertise: Toxicology & Chemistry
Term: 8/26/13 - 6/30/17
Andover, MA 01810
Theodore P. Labuza, Ph.D.
Expertise: Nutrition and Food Science
Term: 7/28/15 - 6/30/19
Morse Alumni Distinguished Professor of Food Science and Engineering
Department of Food and Science and Nutrition,
University of Minnesota
Room 136A Andrew Boss Lab
St. Paul, MN 55108
Michael I. McBurney, Ph.D., F.A.C.N.**
Expertise: Nutrition and Food Science
Term: 7/21/14 - 6/30/18
Vice President
Science, Communication, and Advocacy
DSM Nutritional Products, LLC
45 Waterview Boulevard
Parsippany, NJ 07045
Sharon A. Meyer, Ph.D.
Expertise: Toxicology
Term: 7/21/14 - 6/30/18
Associate Professor
Department of Toxicology
College of Health Sciences, School of Pharmacy
University of Louisiana at Monroe
Monroe, LA 71209
Thomas B. Newman, M.D., M.P.H.
Expertise: Epidemiology, Biostatistics, and Pediatrics
Term: 8/26/15 - 6/30/19
University of California, San Francisco
Department of Epidemiology and Biostatistics
550 16th Street
San Francisco, CA 94143
Urvashi Rangan, Ph.D.*
Expertise: Toxicology and Food Safety
Term: 8/26/13 - 6/30/17
Consumers Union
101 Truman Avenue
Yonkers, NY 10703
A. Catharine Ross, Ph.D.
Expertise: Nutrition
Term: 8/26/13 - 6/30/17
Professor of Nutrition
Occupant, Dorothy Foehr Huck Chair
Department of Nutritional Sciences
Pennsylvania State University
110/204 Chandlee Building
University Park, PA 16802
Dojin Ryu, Ph.D.
Expertise: Food Science and Toxicology
Term: 6/21/2016 6/30/2020
Associate Professor
Bi-State School of Food Science
University of Idaho/Washington State University
875 Perimeter Drive MS 2312
Moscow, ID 83844
Charles Santerre, Ph.D.
Expertise: Toxicology and Food Science
Term: 7/21/14 - 6/30/18
Professor of Food Toxicology
Department of Nutrition
College of Health and Human Sciences
Purdue University
Stone Hall, Rm. 205
700 West State St.
West Lafayette, IN. 47907
James H. Swain, Ph.D., R.D., L.D.
Expertise: Nutrition
Term: 11/26/13 – 6/30/17
Associate Professor & Director
Didactic Program in Dietetics
Department of Nutrition, School of Medicine
Case Western Reserve University
10900 Euclid Avenue
Cleveland, OH 44106
* consumer representatives
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text:
Three weeks ago I had an Extreme Makeover with breast augmentation, rhinoplasty, lower bleph, and some minor liposuction on my chin. I am okay with the results of all the procedures (elated with my breasts, feel good about my bleph) but the rhino was by far the worst part of the experience.
With my new breasts and even the lower bleph, I could see results pretty quickly. Not so with the rhinoplasty! First I had the big cast on my nose and when that came off I looked even worse. Not only did it cause a lot of bruising on my face but my nose looked worse than before. So I called my surgeon in a panic and his nurse told me to wait and be patient and follow the aftercare instructions.
So I did and I am feeling better now. My nose really did reduce in size just like he said, and I kept cleaning out all of the gunk and keeping it tidy like I was supposed to. It took about a month but I finally feel good about the nose job. I no longer have a bump on the top of my nose and the tip is also more defined but not too thin. I have kept the character of my nose and face but also reduced the extremity of the way I looked.
I never doubted that this was what I wanted. Early on in the process that is a decision you have to make. I was worried about the results briefly before I resolved to move forward and deal with it. I am glad I did because not only am I happy but I was a lot less stressed during my recovery. I'm sure that helped me to get better and get to where I am now, one month in (still early) and very happy with what I got! (See my other reviews for thoughts on my other procedures)


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It's Official. I now hate Justin Beiber
I was always sort of on the fence about Justin Beiber. I'm not a fan of his music by any stretch, but he's just a pop artist. Lots of pop music is terrible. But after seeing what Justin Beiber drives, yeah, go ahead and count me in. You can't drive a car painted like that and not be a douche.


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Murder, she wrote
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Eye for detail: Lynda La Plante. Photo: Marco Del Grande
Lynda La Plante's crime stories are making a killing. Angela Bennie reports.
If for nothing else, Lynda La Plante will be remembered as the writer who created one of TV's greatest characters, Detective Inspector Jane Tennison. Vulnerable, canny, unsure, strong, Tennison dominated the Prime Suspect series, giving this hit police show its unique flavour and tension.
In some ways, the middle-aged Tennison was as much the series' subject matter as the stories of crime and punishment.
Admittedly, Helen Mirren had much to do with this. Mirren is a consummate actor, able to capture in one shot Tennison's demeanour of command and contrasting ambivalence. Casting her as Tennison must have been a coup.
"It was the walk," La Plante says bluntly. "I knew she was right when I saw how Helen walked. There's all this up here" - La Plante's hands flutter around the upper body - "with its grace and the fullness. But it's the feet, you see, Helen puts them down hard and almost flat-footed to the ground; they are solid to the earth. I knew she was right for Jane Tennison."
La Plante's remarks are interesting because this is an actor speaking, not a writer. And why not? This multi-award-winning dynamo began professional life as an actor, training at Britain's Royal Academy of Dramatic Art and working with the National Theatre and Royal Shakespeare Company, as well as in TV series such as Minder and The Sweeney.
Boredom with the quality of TV scripts drove La Plante to try her hand at a script. The result was Widows, a gripping yarn about a group of armed female robbers that became a hit TV series and gave La Plante her first BAFTA nomination.
The La Plante we know today - actor, executive producer, writer of novels, film and TV - was on her way. Since then, she has written eight bestsellers, all crime novels, with her ninth, Above Suspicion, launched here last month and climbing to the top of the Herald's bestseller list.
Widows was followed by the now classic Prime Suspect series, which won her a flush of prizes, including six BAFTAs, the Edgar Allen Poe Writer's Award and an Emmy for best miniseries.
Next was The Governor, a drama series about a female prison boss: some episodes were so realistically violent they had to be withdrawn after complaints from viewers.
Exhausted by the Governor experience, but undeterred, La Plante returned to her favourite patch - the meticulously detailed, well-researched police thriller, with a plot filled with twists and double twists, all shaken and stirred by a couple of flawed, multi-dimensional leading characters.
Among her work, the riveting Trial and Retribution is the only real rival to Prime Suspect. The eighth instalment in the series, Suspicion, begins this Friday on the ABC. Lit by a background of incoherence and insanity, increasing tension and brutal murder, Trial and Retribution pits old school against new school: the chemistry goes into slow burn, smoulders a little, then ignites into high drama.
However, be warned - this is not Jane Tennison territory. Times have changed. Detective Chief Inspector Roison Connor is young, blonde and svelte in hip-slung pants and skivvy. La Plante has moved on, and so, she says, has the police department. "There is a new breed in the Met now - they are dressed in Armani suits, they are university-educated, physically fit and very modern women. And they have reached the top. And so my DCI Connor is a university-educated, physically fit, very modern woman. I know my Roison Connor looks like Elle McPherson, but I give her a vile temper. She spits."
La Plante says she is meticulous about detail. "One thing I have learned is that police procedure keeps changing. So before I even begin to write I will spend months in the field."
DCI Connor might be "new school", but gnarled Scottish actor David Hayman, who has played Chief Superintendent Michael Walker throughout the Retribution series, is very much "old school".
"In this I give him his head," says La Plante, who is also executive producer of the series. "The emotional range of this actor is truly impressive. She is new school. He is old school. And old school bends the rules."
Trial and Retribution - Suspicion screens on the ABC over the next two Fridays at 8.30pm.


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Hey there, time traveller!
This article was published 1/6/2012 (1852 days ago), so information in it may no longer be current.
It's not all about you. Some of us never grasp that valuable life lesson, but it's one a close-knit group of Grade 7 students at Windsor School has been grappling with as their rookie season in junior high draws to an end.
Ladies and gentlemen, the Class of 2017 -- whose progress the Free Press is tracking from kindergarten all the way to Grade 12 graduation -- is developing a social conscience.
Their first year in the big leagues at the 238-student, K-8 school in St. Vital has been all about change -- no more recess, no more sitting in the same class all day, and everyone gets their very own locker.
But the biggest change has been the size of the expectations placed on their still-small shoulders. With elementary school a fading memory, it's no longer just about reading, writing and playing with your friends.
It's also about leadership, responsibility and a growing awareness there are other people in the world, people who just might need your help from time to time.
Over the past nine months, the Class of 2017 has helped to organize and execute a wide range of events to help their school, to help their community and to make life better for the poorest of the poor in the slums of East Africa.
With summer vacation knocking at the door, they're throwing themselves into a project close to home -- a drive to raise money to replace the school's decaying wooden play structure, which isn't accessible to children and parents who rely on wheelchairs.
On June 1, the Class of 2017 shepherded the elementary students at Windsor School in a walkathon and a cleanathon to raise money for a new, accessible play structure.
Armed with garbage bags and gloves, the students fanned out in the community to clean up designated areas, then kept a wary eye on the smaller kids as they navigated a circuit around the nearby Canoe Club.
"It's an event that will be good for the community, good for the environment and good for the students because they'll be outside exercising together, cleaning and walking together and helping others," Windsor principal Ann Walker explained in an earlier interview.
"It's not just the school that uses the play structure; it's the whole community because there's not a lot of play structures in the area. We have people in our community who can't use our old play structure, so this is about helping others."
No one needs to tell 12-year-old Griffin how important this latest campaign is -- his younger brother, Tyler, a Grade 1 student, has cerebral palsy.
"My brother uses a wheelchair, and he can't use the play structure because it's surrounded by pea gravel and there's no ramps and it's all stairs and stuff," Griffin quietly tells a visitor. "It feels like he's left out."
Then, looking into the distance, this boy who dreams of playing in the NHL talks proudly of how Tyler's young school buddies show solidarity for their physically challenged friend.
"At recess, kids don't go on the structure," Griffin declares. "They walk around with him. They care!"
Sitting nearby, listening attentively, Aby, 12, eagerly chimes in: "It means a lot to everyone. We all want to help raise money to help Tyler be a part of normal class life. We just want him to feel like he's a normal kid."
For Aby, helping runs in the family. Aided by her brother, Micah, now a Grade 9 student, she created Water From Windsor, an initiative that raises cash to buy water filters to ensure families in a slum area of Nairobi have access to clean water.
In February 2011, spurning a visit to Disneyland, the siblings chose to visit the slums with their family.
"Africa was life-changing," says Aby, who dreams of being a special-needs teacher. "We wanted to do something important and change people's lives. It made me more grateful for everything here, and I made friends."
Sure, the members of the Class of 2017 are still kids, but get them talking about the importance of volunteering and helping others and it becomes clear they're starting to think about things other than hockey and video games.
Noah wasn't necessarily looking forward to riding herd on the elementary school kids during the walkathon -- "They don't listen!" -- but he's proud his class raised about $900 for cancer research during Terry Fox Day events.
"You've gotta help people, and you know you did something. What is it they say? A hero is somebody who does something good, not for a reward but because they can."
With a little prodding, Jesse admits he's trying to lend a hand.
"I help my neighbour clean the snow off her walk just for the good of it," he says, shrugging. "I help my grandparents clean up their house.
"Most kids have a heart."
Speaking of heart, Avery, who is now rocking the new Justin Bieber haircut, was among a handful of classmates at Winnipeg's first-ever rally for We Day last fall, where students were motivated to tackle local and global issues.
"It was basically like a concert, only more inspirational," Avery recalls. "There were tons of speakers talking about how important it is to help people who are less fortunate. When I walked away, I felt I should be doing something like organizing the walkathon and the cleanup."
Replacing the rundown wooden play structure, adds Garrett, is important, "because you're helping the little kids who can't do much and that's good and it's not just for them; it's for future grades, too."
Chris Arnold, one of two homeroom teachers for the Class of 2017, stresses the walkathon/cleanathon is just one of the charitable activities the students have been involved in since starting their career in junior high.
"When they're given an opportunity to see where there's a need, they'll help address it," Arnold says.
"They're still kids, but they're kids who care."
In coming weeks, the students will visit a seniors home, Siloam Mission and Winnipeg Harvest food bank.
"They're among the oldest students in the school and it's their responsibility to lead in a positive way and set a good example for the type of community they want to have," Arnold declares.
For her part, principal Walker agrees a lot more is expected when kids reach Grade 7.
"We don't wait until grade seven and eight to teach about caring for others," she insists, "but there's a big change in grade seven and eight in that they are a lot more involved in the organizing. It's a key time to realize how they can contribute to community service."
Earlier this year, she notes, the kids came up with the idea for a hockey tournament to benefit Water From Windsor. Told to ensure every student was included, the idea grew into a winter festival featuring events revolving around the theme of the earth's water resources.
No one is saying they're angels, but the seventh-graders confess they get a warm, fuzzy feeling helping others.
Sarah recently helped her mom collect cash for breast cancer.
"It feels like something worth doing because other people are sick and you're trying to cure them," she explains.
Julianna volunteers every Thursday afternoon at St. Amant Centre.
"I volunteer at the day care and help little kids," she whispers. "I just have to play with them. It's fun, sometimes."
Naomi is spreading her wings as a babysitter.
"I'm babysitting my cousins and my little brother," she says proudly. "My brother won't listen to me, but my cousins will."
Mackenzie volunteered on pizza day in support of cancer research.
"It's important because you're helping a cause," she points out, "I love helping animals, especially dogs. I hate those commercials where they look so sad.
"Older people think all we do is text each other, but we do more than that."
Hailey couldn't agree more.
"We're the older kids now, and we take on some of the leadership," she advises.
Sydney believes volunteering is important "because if you get a chance to help, you should."
And that's just what Quinn, 13, does in the gym at St. Amant, although he's reluctant to make a big deal of it.
"It makes me feel good that I'm giving back," Quinn admits, thoughtfully.
"When we were young, we just thought about ourselves, but now we have to think about our lives and what path we want to take."
Then, after a brief pause, he adds: "You have to start thinking of what you're going to do in your life."
Danelle Bradshaw, the other homeroom teacher for the Class of 2017, says kids at this age are far more altruistic than adults give them credit for, and they demonstrate true compassion and leadership.
"They're at that age where they don't see the obstacles to things, so they dream really big," Bradshaw says, beaming.
"Sometimes we have to get them focused. They really do stop and think about people's needs. They're at that turning point of being a kid and being an older teen where they can take their own ideas and really run with them."
But, like we said earlier, no one needs to tell that to Griffin, a young man who is working hard to ensure his disabled younger brother can enjoy an accessible play structure at school, just like everyone else.
"This teaches you about life," the flame-haired little philosopher tells a middle-aged visitor.
"This is the early stages of our future. It's when we realize that helping is better than taking."
Here's how you can help
The Class of 2017 is working hard to raise money to replace Windsor School's aging wooden play structure, which isn't accessible to kids and adults who use wheelchairs.
The Windsor Home and School Association, which is leading the campaign, has set a target of $150,000.
If you'd like to help these philanthropic kids, cheques can be made payable to LRSD General Trust (put "Windsor School Play Structure" in the memo line at the bottom left). Tax receipts will be provided for cheques of $10 or more; please send cheques to Windsor School, 80 Cunnington Ave.; Winnipeg, Man., R2M 0W7.