p70 S6 kinase ␣ (p70␣) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70␣ activation in a manner that can be overcome by coexpression of p70␣ with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70␣ polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70␣ phosphorylation in vitro is accompanied by a substantial restoration in p70␣ kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70␣ by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70␣ activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined. p70 S6 kinase ␣ (p70␣), 1 whose major substrate is the 40 S ribosomal protein S6, plays a critical role in the translation of a subclass of mRNAs that contain a short oligopyrimidine sequence immediately following the transcriptional start site (1). p70␣ is activated in response to insulin/mitogens in vivo through a multisite phosphorylation of serine and threonine residues (2). Several sets of independently regulated p70␣ phosphorylation sites have been identified (3-6); one set consists of Ser/Thr-Pro motifs, five of which are clustered in a psuedosubstrate autoinhibitory domain in the noncatalytic carboxyl-terminal tail (Ser-434, Ser-441, Ser-447, Ser-452, and Thr-444 in p70␣), and two others, Thr-390 and Ser-394, are located in a 65-amino acid segment immediately carboxyl-terminal to the kinase catalytic domain. A second set of regulated phosphorylation sites, Thr-412 and Ser-427, exhibit the sequence motif Phe-Ser/Thr-Phe/Tyr. Thr-252, located on the activation loop in catalytic subdomain VIII, is the site at which 3-phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates p70␣ (7,8). Among these, the phosphorylation of Thr-252, Ser-394, and Thr-412 is necessary for the activation of p70␣ kinase catalytic function; the attainment of physiologic levels of p70␣ activity results from a strongly synergistic, positive site-site interaction between the phosphorylated Thr-252 and Thr-412 residues (7).In addition to its regulation by insulin and mitogens through PI-3 kinase-dependent pathways, p70␣ can also be activated by increasing concentrations of extracellular amino acids in the absence of serum or mitogens to the level attained by maximal mitogen stimulation (9-11). Moreover, a thr...

Presented herein is the initial clinical experience with 3D printing to facilitate patient's pre-surgical understanding of their kidney tumor and surgery.

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Our results indicated that isoflavones and curcumin could modulate serum PSA levels. Curcumin presumably synergizes with isoflavones to suppress PSA production in prostate cells through the anti-androgen effects.

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BackgroundProstate cancer cells communicate reciprocally with the stromal cells surrounding them, inside the prostate, and after metastasis, within the bone. Each tissue secretes factors for interpretation by the other. One stromally-derived factor, Hepatocyte Growth Factor (HGF), was found twenty years ago to regulate invasion and growth of carcinoma cells. Working with the LNCaP prostate cancer progression model, we found that these cells could respond to HGF stimulation, even in the absence of Met, the only known HGF receptor. The new HGF binding partner we find on the cell surface may help to clarify conflicts in the past literature about Met expression and HGF response in cancer cells.MethodsWe searched for Met or any HGF binding partner on the cells of the PC3 and LNCaP prostate cancer cell models, using HGF immobilized on agarose beads. By using mass spectrometry analyses and sequencing we have identified nucleolin protein as a novel HGF binding partner. Antibodies against nucleolin (or HGF) were able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. Western blots, RT-PCR, and immunohistochemistry were used to assess nucleolin levels during prostate cancer progression in both LNCaP and PC3 models.ResultsWe have identified HGF as a major signaling component of prostate stromal-conditioned media (SCM) and have implicated the protein nucleolin in HGF signal reception by the LNCaP model prostate cancer cells. Antibodies that silence either HGF (in SCM) or nucleolin (on the cell surfaces) eliminate the adhesion-stimulatory effects of the SCM. Likewise, addition of purified HGF to control media mimics the action of SCM. C4-2, an LNCaP lineage-derived, androgen-independent human prostate cancer cell line, responds to HGF in a concentration-dependent manner by increasing its adhesion and reducing its migration on laminin substratum. These HGF effects are not due to shifts in the expression levels of laminin-binding integrins, nor can they be linked to expression of the known HGF receptor Met, as neither LNCaP nor clonally-derived C4-2 sub-line contain any detectable Met protein. Even in the absence of Met, small GTPases are activated, linking HGF stimulation to membrane protrusion and integrin activation. Membrane-localized nucelolin levels increase during cancer progression, as modeled by both the PC3 and LNCaP prostate cancer progression cell lines.ConclusionWe propose that cell surface localized nucleolin protein may function in these cells as a novel HGF receptor. Membrane localized nucleolin binds heparin-bound growth factors (including HGF) and appears upregulated during prostate cancer progression. Antibodies against nucleolin are able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. HGF-nucleolin interactions could be partially responsible for the complexity of HGF responses and met expression reported in the literature.

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