RPS6 phosphorylation occurs to a greater extent in the periphery of human skeletal muscle fibers, near focal adhesions, after anabolic stimuli.

Following anabolic stimuli (mechanical loading and/or amino acid provision), the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1-mediated phosphorylation events occur in these peripheral regions or before translocation (i.e., in central regions). We therefore aimed to determine the cellular location of a mTORC1-mediated phosphorylation event, RPS6Ser240/244, in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25 g/kg protein and 0.75 g/kg carbohydrate) alone [n = 7; 23 ± 5 yr; 76.8 ± 3.6 kg; and 13.6 ± 3.8% body fat (BF), FED] or following a whole body resistance exercise bout (n = 7; 22 ± 2 yr; 78.1 ± 3.6 kg; and 12.2 ± 4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300 min following anabolic stimuli. RPS6Ser240/244 phosphorylation measured by immunofluorescent staining or immunoblot was positively correlated (r = 0.76, P < 0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120 min (∼54% and ∼138%, respectively, P < 0.05) but was greater in EXFED at both poststimuli time points (P < 0.05). The peripheral-to-central ratio of p-RPS6240/244 staining displayed a similar pattern, even when corrected for total RPS6 distribution, suggesting RPS6 phosphorylation occurs to a greater extent in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120 min irrespective of stimulus (P = 0.006) before returning to PRE at 300 min. These data confirm that RPS6Ser240/244 phosphorylation occurs in the region of human muscle fibers to which mTOR translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 regulation in vivo.

Myotube Protein Content Associates with Intracellular L-Glutamine Levels.

BACKGROUND/AIMS: Skeletal mass loss is reported in several catabolic conditions and it has been associated with a reduced intracellular L-glutamine content. We investigated the association of intracellular L-glutamine concentration with the protein content in skeletal muscle cells. METHODS: We cultivated C2C12 myotubes in the absence or presence of 2 (reference condition), 8 or 16 mM L-glutamine for 48 hours, and the variations in the contents of amino acids and proteins measured. We used an inhibitor of L-glutamine synthesis (L-methionine sulfoximine - MSO) to promote a further reduction in intracellular L-glutamine levels. Amino acids contents in cells and media were measured using LC-MS/MS. We measured changes in phosphorylated Akt, RP-S6, and 4E-BP1contents in the absence or presence of insulin by western blotting. RESULTS: Reduced intracellular L-glutamine concentration was associated with decreased protein content and increased protein breakdown. Low intracellular glutamine levels were also associated with decreased p-Akt contents in the presence of insulin. A further decrease in intracellular L-glutamine caused by glutamine synthetase inhibitor reduced protein content and levels of amino acids generated from glutamine metabolism and increased bAib still further. Cells exposed to high medium glutamine levels did not have any change in protein content but exhibited increased contents of the amino acids derived from L-glutamine metabolism. CONCLUSION: Intracellular L-glutamine levels per se play a role in the control of protein content in skeletal muscle myotubes.

Clinical Outcomes of Isolated Regional Lymph Node Recurrence in Patients With Malignant Cutaneous Melanoma.

BACKGROUND/AIM: Regional lymph node recurrence (RLNR) is the most common pattern of recurrence within 2 years from the diagnosis of patients with non-metastatic malignant cutaneous melanoma. However, isolated RLNR without distant metastasis has been rarely studied. PATIENTS AND METHODS: Forty patients with isolated RLNR as a first recurrence were analyzed retrospectively. The clinical outcomes and prognostic impact of clinicopathologic parameters were analyzed. Immunostaining for FOXP3, VEGF, pAKT, and pS6 was also performed. RESULTS: The median disease-free interval from first diagnosis to isolated RLNR and post-recurrence recurrence-free survival (pRFS) were 12 months and 7.2 months, respectively. Distant failure was the most common pattern of failure after isolated RLNR (67.5%). The number of initially harvested lymph nodes (LN) >7 and LN ratio >22.2% at the time of recurrence were prognosticators for pRFS in multivariate analysis. None of the tested biomarkers were significantly related to prognosis. The 5-year post-recurrence overall survival rate was 84.9%. CONCLUSION: Most patients with isolated RLNR will experience a second failure within months, especially distantly. The number of initially harvested LNs and LN ratio at the time of recurrence could predict pRFS.

mTOR, VEGF, PDGFR, and c-kit signaling pathway activation in Kaposi sarcoma.

Kaposi sarcoma (KS) is a locally progressive, intermediate-grade vascular neoplasm with no known cure, high recurrence rates, and potential for wide dissemination. Low efficacy and high toxicity limit current therapeutic options for advanced disease. Activation of mammalian target of rapamycin (mTOR), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and c-kit signaling pathways has been implicated in KS pathogenesis and may suggest a role for targeted inhibitors. KS cases were retrospectively retrieved (N=274), most (90%) associated with human immunodeficiency virus. Tissue microarray slides were stained with human herpes virus-8, Friend leukemia integration 1 transcription factor, CD117 (c-kit), phospho-S6 (pS6), PDGF receptor-ß, VEGF, and phospho-mTOR. Both intensity and extent of staining were scored. Multiplying these scores for each core yielded total staining H-scores. Human herpes virus-8 was positive in 87% and Friend leukemia integration 1 transcription factor in 95.7% of cases. Most were also VEGF+ (97.6%), pS6+ (95.7%), CD117+ (92.5%), and PDGFRB+ (87.4%). Approximately half (55.6%) were phospho-mTOR+. There was no significant difference in staining among patients with low (<500 cells/mm3) or preserved CD4 T-cell counts. Immunohistochemistry confirms upregulation of the mTOR, PDGF, VEGF, and c-kit pathways in a large cohort of KS samples. Of proteins tested, pS6, downstream of mTOR, demonstrated the highest proportion of strong positivity (67.1%). These results support the possibility of using targeted inhibitors in KS. Overexpression was independent of CD4 count, suggesting that even patients with low counts may be targeted therapy candidates.

Improvement of tuberous sclerosis complex (TSC) skin tumors during long-term treatment with oral sirolimus.

BACKGROUND: Oral mechanistic target of rapamycin inhibitors have been shown to reduce visceral tumor volume in patients with tuberous sclerosis complex (TSC). OBJECTIVE: We sought to evaluate the cutaneous response to oral sirolimus in patients with TSC and an indication for systemic treatment, including long-term effects. METHODS: A retrospective analysis of 14 adult patients with TSC prescribed sirolimus to treat lymphangioleiomyomatosis was performed. Serial photographs of angiofibromas, shagreen patches, and ungual fibromas taken before, during, and after the treatment period were blinded, then assessed using the Physician Global Assessment of Clinical Condition (PGA). Microscopic and molecular studies were performed on skin tumors harvested before and during treatment. RESULTS: Sirolimus significantly improved angiofibromas (median treatment duration 12 months; median PGA score 4.5 [range 1.5-5]; Wilcoxon signed rank test, P = .018) and shagreen patches (median treatment duration 10 months; median PGA score 4.5 [range 3.5-5]; Wilcoxon signed rank test, P = .039), whereas ungual fibromas improved in some patients (median treatment duration 6.5 months; median PGA score 4.66 [range 2.75-5]; Wilcoxon signed rank test, P = .109). Clinical, immunohistochemical, or molecular evidence of resistance was not observed (range 5-64 months of treatment). LIMITATIONS: This was a retrospective analysis limited to adult women with lymphangioleiomyomatosis. CONCLUSION: Oral sirolimus is an effective long-term therapy for TSC skin tumors, particularly angiofibromas, in patients for whom systemic treatment is indicated.

Suprasellar chordoid neoplasm with expression of thyroid transcription factor 1: evidence that chordoid glioma of the third ventricle and pituicytoma may form part of a spectrum of lineage-related tumors of the basal forebrain.

Chordoid glioma of the third ventricle is a rare neuroepithelial tumor characterized by a unique histomorphology and exclusive association with the suprasellar/third ventricular compartment. Variously interpreted as either astrocytic- or ependymal-like, and speculatively ascribed to the lamina terminalis/subcommissural organ, its histogenesis remains, nevertheless, unsettled. Here, we report on a suprasellar chordoid glioma occurring in a 52-year-old man. Although displaying otherwise typical morphological features, the tumor was notable for expression of thyroid transcription factor 1, a marker of tumors of pituicytic origin in the context of the sellar region. We furthermore found overlapping immunoprofiles of this example of chordoid glioma and pituicytic tumors (pituicytoma and spindle cell oncocytoma), respectively. Specifically, phosphorylated ribosomal protein S6, a marker of mTOR pathway activation, was expressed in both groups. Based on these findings, we suggest that chordoid glioma and pituicytic tumors may form part of a spectrum of lineage-related neoplasms of the basal forebrain.

Extinction, applied after retrieval of auditory fear memory, selectively increases zinc-finger protein 268 and phosphorylated ribosomal protein S6 expression in prefrontal cortex and lateral amygdala.

Retrieval of consolidated memories induces a labile phase during which memory can be disrupted or updated through a reconsolidation process. A central component of behavioral updating during reconsolidation using a retrieval-extinction manipulation (Ret+Ext) is the synaptic removal of a calcium-permeable-α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (CP-AMPARs) in the lateral amygdala-a metabotropic GluR1 receptor (mGluR1) dependent mechanism. In the present study, we investigate the effect of Ret+Ext on the expression of molecular markers that could play a role in the reconsolidation process. Specifically, we tested the effects of Ret+Ext on the global expression of zinc-finger 268 protein (Zif268), a marker previously found to be implicated in memory reconsolidation, to confirm its occurrence after retrieval (Ret) and Ret+Ext. We also evaluated the global expression of phosphorylated ribosomal protein S6 (rpS6P), here proposed as a marker of the mGluR1-mediated memory process induced by Ret+Ext. The expression of both markers (zif268, rpS6P) was assessed by immunolocalization in prelimbic cortex (PRL), infralimbic cortex (IL), ventral subdivision of the lateral amygdala (LA) and hippocampus CA1 (CA1) in fear-conditioned rats. Our results showed that retrieval and Ret+Ext, but not extinction alone, increased Zif268 expression in prefrontal cortex and lateral amygdala. Ret+Ext, but not retrieval, retrieval followed by context exposure or extinction alone, increased the expression of rpS6P in prefrontal cortex and LA. In summary, (i) Zif268 increased after retrieval confirming that reconsolidation is engaged in our conditions, (ii) Zif268 increased after Ret+Ext confirming that it does not simply reflect an extinction or reconsolidation disruption (Zif268 level of expression should be lower in both cases) and (iii) rpS6P increased after Ret+Ext, but not after extinction, suggesting, as expected, a potential mGluR1 mediated molecular mechanism specific for Ret+Ext. Together with the Zif268 increase, our results suggest that the Ret+Ext induced memory process is more similar to reconsolidation updating than extinction facilitation.

Identification of proteomic biomarkers predicting prostate cancer aggressiveness and lethality despite biopsy-sampling error.

BACKGROUND: Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. METHODS: Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a 'high' and a 'low' tumour microarray, respectively. RESULTS: Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. CONCLUSIONS: Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness.

Phosphorylated S6 as an immunohistochemical biomarker of vulvar intraepithelial neoplasia.

As life expectancy lengthens, cases of non-viral-associated vulvar squamous cell carcinoma and its precursor lesion, so-called differentiated vulvar intraepithelial neoplasia (VIN), continue to increase in frequency. Differentiated VIN often is difficult to recognize and failure to detect it before invasion results in morbidity and mortality. Thus, identification of a reliable biomarker for this type of lesion would be of great clinical benefit. Our recent studies have identified activation (ser235/236 phosphorylation) of ribosomal protein S6 (p-S6) in basal epithelial cells as an event that precedes and accompanies laminin γ(2) overexpression in most preinvasive oral dysplasias. To test this as a potential biomarker of vulvar dysplasia, we immunostained seven differentiated VINs and nine papillomavirus-related 'classic' VINs, most of which were associated with carcinoma, for p-S6. All carcinomas, all differentiated VINs, and most classic VINs contained regions of p-S6 staining in the basal layer, whereas basal and parabasal cells of normal vulvar epithelium and hyperplastic and inflamed lesions lacking cellular atypia were p-S6 negative. Laminin γ(2) was expressed in a subset of VINs, always occurring within basal p-S6 positive regions, as we had found previously for oral dysplasias. Lichen sclerosus is considered a potential precursor of vulvar carcinoma. Two lichen sclerosus lesions of patients with a concurrent carcinoma and one of six lichen sclerosus lesions without atypia or known concurrent carcinoma were basal p-S6 positive. In summary, there is a distinct difference in p-S6 basal cell layer staining between benign and neoplastic vulvar squamous epithelium, with consistent staining of differentiated VIN and of some lichen sclerosus lesions. These results support further studies to assess the potential of p-S6 as a biomarker to identify vulvar lesions at risk of progressing to invasive cancer.

Evidence of ambiguous differentiation and mTOR pathway dysregulation in subependymal giant cell astrocytoma.

OBJECTIVE: The exact cell of origin of subependymal giant cell astrocytoma is debated but most currently consider the tumor in the astrocytic category. Mutations and subsequent biallelic inactivation of TSC1 encoding hamartin, or TSC2 encoding tuberin appear to be the underlying genetic aberrations. Inactivation leads to loss of proteins that inhibit mammalian target of rapamycin (mTOR ) disrupting tightly regulated cell functions. MATERIAL AND METHOD: We analyzed the expression of tuberin and hamartin along with an array of neuroepithelial markers in 9 subependymal giant cell astrocytomas. In addition, RPS6 and 4EBP1 regulatory proteins that are downstream in the mTOR pathway were also evaluated. RESULTS: While hamartin and tuberin expression levels were relatively decreased compared to control tissue, this was not of particular practical use to detect the mutated gene since low levels of positivity could be detected throughout the central nervous system. As expected, the levels of RPS6 and 4EBP1 were increased, further confirming the activation of the mTOR pathway. GFAP was positive in 5 cases, while Synaptophysin positivity was found in all tumors. CD34 (a marker often observed in well differentiated glio-neuronal tumors), Olig2 (a nuclear marker present in most gliomas), IDH1 and IDH2 were entirely negative in all tumor cells. Ki67 (MIB-1) showed a low proliferation rate ranging from 2% to 8%. CONCLUSION: Staining with neuroepithelial markers supports the suggestion of ambiguous differentiation. Subependymal giant cell astrocytomas do not appear to have the typical expression profiles of astrocytic tumors, under which they have been classified.

Mitotic lymphoma cells are characterized by high expression of phosphorylated ribosomal S6 protein.

Growth factors and mitogens influence signaling pathways and often induce the activity of p70S6 kinase (p70S6K), which in turn phosphorylates the ribosomal S6 protein (S6). Although recent data are rather conflicting, the overall view suggests that phosphorylated S6 is a regulator of global protein synthesis, cell proliferation, cell size and glucose homeostasis. In the present work, emphasis was given to cell cycle-dependent activation of S6 focusing mainly on human lymphoid and lymphoma cells. Paraffin-embedded human tissue blocks from lymph node and different tumor biopsies as well as in vitro cell lines were investigated by immunohistochemistry, immunocytochemistry, flow cytometry and Western blotting using antibodies directed against phospho-S6, phospho-mTOR, phospho-p70S6K and phospho-Histone H3. To enrich the cell number in different phases of the cell cycle, nocodazole, staurosporine or rapamycin were used in cell cultures. We observed strong phospho-S6 positivity by immunostainings in the dividing lymphoid cells of reactive lymph nodes and in lymphoma cells cultured in vitro. Phospho-S6 protein levels were shown to be elevated throughout mitosis in lymphoma cells; however, the high expression of phospho-S6 in mitotic cells was not a general hallmark of tumor cell types studied so far: phospho-S6-negative mitotic cells were detected in several carcinoma and sarcoma biopsies. These observations may have practical implications as they raise the possibility to consider p70S6K and/or S6 as a potential therapeutic target-besides mTOR-in certain lymphomas and perhaps in clinical immunosuppression.

Immunohistochemical assessment of signal transduction and cell-cycle networks in neural tumors.

The ability to detect transient changes in molecular networks lies at the heart of cancer biology research. This is especially apparent during tumorigenesis, where initiating mutations typically affect mitogens and cell-cycle molecules such as PDGF or retinoblastoma protein (Rb). One of the primary consequences of such processes is the inappropriate stimulation of downstream targets, normally through posttranslational modification. Immunohistochemistry (IHC) provides an important tool for assessing such changes in situ, permitting different aspects of tumor biology to be examined as a tissue undergoes transformation. Nevertheless, this can be difficult to achieve, particularly in complex environments like the brain. Here, we provide the automated methodology we have employed for the successful detection of phosphorylation of S6 ribosomal protein (S6-RP) and the retinoblastoma protein (Rb) in response to PDGF stimulation in a mouse model of glial brain tumor development.

Ribosomal protein S6 is highly expressed in non-Hodgkin lymphoma and associates with mRNA containing a 5' terminal oligopyrimidine tract.

The molecular mechanism(s) linking tumorigenesis and morphological alterations in the nucleolus are presently coming into focus. The nucleolus is the cellular organelle in which the formation of ribosomal subunits occurs. Ribosomal biogenesis occurs through the transcription of ribosomal RNA (rRNA), rRNA processing and production of ribosomal proteins. An error in any of these processes may lead to deregulated cellular translation, evident in multiple cancers and 'ribosomopathies'. Deregulated protein synthesis may be achieved through the overexpression of ribosomal proteins as seen in primary leukemic blasts with elevated levels of ribosomal proteins S11 and S14. In this study, we demonstrate that ribosomal protein S6 (RPS6) is highly expressed in primary diffuse large B-cell lymphoma (DLBCL) samples. Genetic modulation of RPS6 protein levels with specifically targeted short hairpin RNA (shRNA) lentiviruses led to a decrease in the actively proliferating population of cells compared with control shRNA. Low-dose rapamycin treatments have been shown to affect the translation of 5' terminal oligopyrimidine (5' TOP) tract mRNA, which encodes the translational machinery, implicating RPS6 in 5' TOP translation. Recently, it was shown that disruption of 40S ribosomal biogenesis through specific small inhibitory RNA knockdown of RPS6 defined RPS6 as a critical regulator of 5' TOP translation. For the first time, we show that RPS6 associates with multiple mRNAs containing a 5' TOP tract. These findings expand our understanding of the mechanism(s) involved in ribosomal biogenesis and deregulated protein synthesis in DLBCL.

Utility of lyophilized PMA and ionomycin to stimulate lymphocytes in whole blood for immunological assays.

BACKGROUND: The need to implement robust biomarkers in clinical trials has never been greater, and such efforts can be easily compromised by reagent instability or simple human error during assay set-up. Many biotechnology and pharmaceutical companies are introducing efforts to conduct biomarker studies under more rigorous settings, and the use of plates or tubes pre-loaded with stimulation or staining reagents could be of value for studies that involve flow cytometry. METHODS: Five reagents lyophilized from ethanol or CHAPS buffer stock solution of phorbol 12-myristate 13-acetate (PMA) and ionomycin were benchmarked against standard DMSO liquid formulation for their stimulation equivalency. The median fluorescence intensity of phosphorylated ribosomal protein S6 in lymphocytes was assessed on a BD FACSCalibur. RESULTS: We demonstrate here that tubes pre-loaded with lyophilized versions of the liquid reagents can provide equivalent stimulation in healthy volunteer specimens. CONCLUSIONS: The value of this approach is that it safeguards against omission or erroneous addition of bulk liquid formulations of PMA and ionomycin to the reaction vessel (i.e., plate or tube) and also lends itself to extended stability/shelf-life of these reagents. On the basis of this initial success, we plan to expand our evaluation of lyophilized reagents so that they can be incorporated into our clinical biomarker campaigns as appropriate.

Activation of microglia/macrophages expressing phosphorylated S6 ribosomal protein in a case of hemimegalencephaly with progressive calcification and atrophy.

A 3-year-old boy with right hemimegalencephaly (HME) showed massive calcification in the subcortical white matter and progressive atrophy of the affected hemisphere. Hemispherotomy was successful in amelioration of the patient's intractable epilepsy, and a surgical specimen from the epileptic focus was examined pathologically. Disarrangement of cortical layers along with dysmorphic appearance of neurons, balloon cells in the cortex and white matter, bi-layered calcifications in the superficial cortical layer and subcortical white matter, heterotopic neurons in the white matter, and diffuse astrogliosis were noted. Perivascular clustering of alpha-B-crystallin positive balloon cells was occasionally observed in the area of calcification. A diffuse increase was observed in the number of CD68-positive microglia/macrophages, particularly in perivascular and peri-calcification areas. These cells were often located within the calcification foci, which implicates their participation in the calcification process. Phosphorylated S6 ribosomal protein (P-S6) was expressed in large-sized neurons and numerous balloon cells, as well as in CD68-positive cells. In contrast, phosphorylated mammalian target of rapamycin (mTOR) was expressed in a small percentage of astrocytes, and phosphorylated p70S6 kinase was rarely identified in perivascular cells. These findings suggest that inflammatory processes have contributed to the pathogenesis of progressive calcification and atrophy in the megalencephalic hemisphere in this patient. Dissociation of expression of mTOR cascade components is common to other reported cases of HME, but P-S6 expression in microglia/macrophages has not been recognized. The cellular mechanism and significance of P-S6-specific activation of the mTOR cascade in HME, particularly in the inflammatory cell lineage, should be explored further.

Phospho-S6 ribosomal protein: a potential new predictive sarcoma marker for targeted mTOR therapy.

Metastatic sarcomas are commonly resistant to chemotherapy. The serine/threonine kinase, mammalian target of rapamycin (mTOR), is a protein kinase of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway thought to have a key role in controlling cancer growth and thus is an important target for cancer therapy. Several inhibitors of mTOR are in clinical trials, including AP23573, which is being tested on metastatic sarcomas and other tumors. We hypothesized that a marker for the activity of mTOR, phosphorylated S6 ribosomal protein, would be predictive of clinical response to the drug, that is, high tumor expression would signify better response than low expression. This was a blinded study. Of 26 patients treated, 20 remained on study, with available paraffin blocks. Fourteen patients received AP23573 alone and six patients received AP23573 in combination with adriamycin. An antibody to the phosphorylated S6 ribosomal protein was used to stain the tumors, all high-grade sarcomas. Pretreatment biopsy or resection material was tested: the original tumor (n=6) or tumor recurrence/metastasis (n=14); either of these may have been after treatment with other agents. Staining was scored for both quantity/percentage of tumor cells and intensity. Scoring was performed without knowledge of tumor response. Staining quantity could be categorized into two natural groups: high expressors (> or =20% of tumor cells, 11 cases) and low expressors (0-10% of tumor cells, 9 cases). The high-expression group had eight stable and three progressive cases (73% stable disease); the low-expression group had three stable and six progressive cases (67% progressive disease). Chi-square analysis showed statistical significance (P< or =0.05) at this initial cutoff (10%) selected blindly. The level of phosphorylated S6 ribosomal protein expression was predictive of early tumor response to the mTOR inhibitor, suggesting that this is a promising new predictive sarcoma marker for targeted mTOR inhibitor therapy.

Phosphorylation profiles of protein kinases in alveolar and embryonal rhabdomyosarcoma.

Rhabdomyosarcoma is the most common pediatric soft-tissue sarcoma, which includes two major subtypes, alveolar and embryonal rhabdomyosarcoma. The mechanism of its oncogenesis is largely unknown. However, the oncogenic process of rhabdomyosarcoma involves multi-stages of signaling protein dysregulation characterized by prolonged activation of tyrosine and serine/threonine kinases. To better understand this protein dysregulation, we evaluated the phosphorylation profiles of multiple tyrosine and serine/threonine kinases to identify whether these protein kinases are activated in rhabdomyosarcoma. We applied immunohistochemistry with phospho-specific antibodies to examine phosphorylation levels of selected receptor and non-receptor tyrosine kinases, mammalian target of rapamycin (mTOR), p70S6K, and protein kinase C (PKC) isozymes on alveolar and embryonal rhabdomyosarcoma tissue microarray slides. Our results showed that the phosphorylation levels of these kinases are elevated in some rhabdomyosarcoma tissues compared to normal tissues. Phosphorylation levels of receptor and non-receptor tyrosine kinases are elevated between 26 and 68% in alveolar rhabdomyosarcoma and between 24 and 71% in embryonal rhabdomyosarcoma, respectively, compared to normal tissues. In addition, phosphorylation levels of mTOR and its downstream targets, p70S6K, S6, and 4EBP1, are increased between 50 and 72% in both subtypes of rhabdomyosarcoma. Further, phosphorylation levels of PKCalpha, PKCdelta, PKCtheta, and PKCzeta/lambda are upregulated between 57 and 69% in alveolar rhabdomyosarcoma and between 43 and 72% in embryonal rhabdomyosarcoma. This is the first report to create a phosphorylation profile of tyrosine and serine/threonine kinases involved in the mTOR and PKC pathways of alveolar and embryonal rhabdomyosarcoma. These protein kinases may play roles in the development or tumor progression of rhabdomyosarcomas and thus may serve as novel targets for therapeutic intervention.

The selective protein kinase C beta inhibitor enzastaurin induces apoptosis in cutaneous T-cell lymphoma cell lines through the AKT pathway.

Enzastaurin displays pro-apoptotic properties against a spectrum of malignancies and is currently being investigated in clinical trials. We have investigated the effects of enzastaurin on the viability of the cutaneous T-cell lymphoma cell lines HuT-78 and HH by using 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, cell cycle analysis, propidium iodide and annexin-V staining, and caspase-3-mediated proteolytic activation. Enzastaurin-treatment decreased cell viability, increased annexin V-FITC-positive cells, and increased the proportion of sub-G1 populations in both cell lines that was not reversed by the T-cell growth stimulating cytokines IL-2, IL-7, IL-15. Enzastaurin-induced cell death involved caspase-3-activated cleavage of poly(ADP-ribose) polymerase that was inhibited by the pan-caspase inhibitor ZVAD-fmk, whereas the increase in sub-G1 population was only partially inhibited by ZVAD-fmk. Furthermore, enzastaurin downregulated AKT activity and its downstream effectors GSK3beta and ribosomal protein S6. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been implicated in the growth and survival of hematologic malignancies and inhibition of this pathway is considered as a therapeutic target. Protein kinase C activation contributes to PI3K/AKT activation, but it is unknown how enzastaurin may interfere with signaling through this pathway. These results demonstrate that enzastaurin, at clinically achievable concentrations, induces apoptosis and affects AKT signaling, and provide a rationale for further in vivo studies addressing the therapeutic efficacy in cutaneous T-cell lymphoma patients.

Molecular dynamics of a protein surface: ion-residues interactions.

Time-resolved measurements indicated that protons could propagate on the surface of a protein or a membrane by a special mechanism that enhanced the shuttle of the proton toward a specific site. It was proposed that a suitable location of residues on the surface contributes to the proton shuttling function. In this study, this notion was further investigated by the use of molecular dynamics simulations, where Na(+) and Cl(-) are the ions under study, thus avoiding the necessity for quantum mechanical calculations. Molecular dynamics simulations were carried out using as a model a few Na(+) and Cl(-) ions enclosed in a fully hydrated simulation box with a small globular protein (the S6 of the bacterial ribosome). Three independent 10-ns-long simulations indicated that the ions and the protein's surface were in equilibrium, with rapid passage of the ions between the protein's surface and the bulk. However, it was noted that close to some domains the ions extended their duration near the surface, thus suggesting that the local electrostatic potential hindered their diffusion to the bulk. During the time frame in which the ions were detained next to the surface, they could rapidly shuttle between various attractor sites located under the electrostatic umbrella. Statistical analysis of the molecular dynamics and electrostatic potential/entropy consideration indicated that the detainment state is an energetic compromise between attractive forces and entropy of dilution. The similarity between the motion of free ions next to a protein and the proton transfer on the protein's surface are discussed.

Nutrient restriction differentially modulates the mammalian target of rapamycin signaling and the ubiquitin-proteasome system in skeletal muscle of cows and their fetuses.

The mammalian target of rapamycin (mTOR) signaling controls nutrient-stimulated protein synthesis in skeletal muscle, whereas ubiquitin-proteasome systems control the degradation of myofibrillar proteins. The objective of this study was to elucidate the effect of nutrient restriction on the mTOR signaling and ubiquitin-proteasome system in the skeletal muscle of cows and their fetuses. Beginning 30 d after conception, 20 cows were fed either a control diet that provided 100% nutrient requirements or a nutrient-restricted diet at 68.1% of NE(m) and 86.7% of metabolizable protein requirement. Cows were slaughtered on 125 d of gestation, and the LM of both cows and fetuses was sampled for the measurement of mTOR, ribosomal protein S6, adenosine 5'-monophosphate-activated protein kinase (AMPK), and protein ubiquitylation. When comparing the muscle samples from nutrient-restricted and control cows and their fetuses, no difference was observed for the content of mTOR and ribosomal protein S6, but the phosphorylation of mTOR at Ser(2448) and ribosomal protein S6 at Ser(235/336) were greater (P < 0.05) in control muscle than in muscle from nutrient-restricted animals. Because the phosphorylation of mTOR and ribosomal protein S6 upregulates translation, these results showed that nutrient restriction inhibits protein synthesis in muscle. The activity of AMPK in the muscle of nutrient-restricted cows was significantly lower (P = 0.05) than that of control cows. The protein ubiquitylation, however, was greater (P < 0.05) in the muscle from nutrient-restricted cows, showing accelerated protein degradation. No difference in the protein ubiquitylation was detected for fetal muscle. Data suggested that the decreased protein synthesis and promoted protein degradation resulted in muscle atrophy of pregnant cows, but not in fetal muscle. Results of this study show that in response to nutrient restriction, protein degradation was differentially regulated between cow and fetal muscle. The atrophy of cow muscle during nutrient deficiency may involve the enhanced degradation of muscle proteins.