S6 Activity Research Articles

Effects of epidermal growth factor receptor and phosphatase and tensin homologue gene expression on the inhibition of U87MG glioblastoma cell proliferation induced by protein kinase inhibitors

The aim of the present study was to analyse the antiproliferative effects and mechanisms of action of protein kinase inhibitors (PKIs) in human glioblastoma multiforme (GBM) cells with different epidermal growth factor receptor (EGFR) and phosphatase and tensin homologue (PTEN) status. The GBM cell models were established by transfection of plasmids carrying wild-type EGFR, mutated EGFRvIII or PTEN and clonal selection in U87MG cells. Phosphatidylinositol 3-kinase (PI3-K)/AKT pathway-focused gene profiles were examined by real-time polymerase chain reaction-based assays, protein expression was evaluated by western blotting and the antiproliferative effects of PKI treatment were determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in GBM cells. The cell model with intact PTEN and low EGFR levels was the most sensitive to treatment with the EGFR inhibitor erlotinib, whereas the model with EGFRvIII was the most resistant to treatment with the mitogen-activated protein kinase kinase inhibitor U0126. The dual PI3-K and mammalian target of rapamycin (mTOR) inhibitor PI103 had the most potent antiproliferative effects against all GBM cells tested. Following simultaneous stimulation of AKT and extracellular signal-regulated kinase, rapamycin concentrations > 0.5 nmol/L failed to exhibit a further growth inhibitory effect. Concurrent inhibition of mTOR and ribosomal protein s6 activity may underlie the inhibition of GBM proliferation by PKI. In conclusion, overexpression of EGFR or EGFRvIII, accompanied by a loss of PTEN, contributed to the activation of multiple intracellular signalling pathways in GBM cells. Rigorous examination of biomarkers in tumour tissues before and after treatment may be necessary to determine the efficacy of PKI therapy in patients with GBM.

Effects of Ponatinib and Other Novel TKI On Growth, Survival, and Function of Neoplastic Eosinophils Carrying FIP1L1/Pdgfra

AbstractAbstract 1760In chronic eosinophilic leukemia (CEL), the transforming oncoprotein FIP1L1-PDGFRA (F/P) is a major target of therapy. In most patients, the PDGFRA-targeting tyrosine kinase inhibitor (TKI) imatinib induces complete and durable molecular remissions. For patients who are intolerant or resistant against imatinib, novel TKI may serve as potential alternative therapy. Indeed, several different TKI have been described to act on Ba/F3 cells transfected with F/P, and some even block the activity of imatinib-resistant F/P mutants. However, little is known about the effects of novel TKI on growth and survival of primary neoplastic eosinophils. In the current study, we examined the in vitro effects of 12 kinase blockers on growth and viability as well as cytokine-induced migration of EOL-1 cells, a human F/P+ eosinophil leukemia cell line. In addition, we examined TKI effects on primary human neoplastic eosinophils obtained from a patient with F/P+ CEL, one with aggressive systemic mastocytosis and massive eosinophilia (ASM-eo) and one with reactive hypereosinophilia (HE). In EOL-1 cells, major growth-inhibitory effects were seen with all PDGFRA-blocking agents, with IC50 values in the low nM-range: ponatinib: 0.1–0.2 nM, sorafenib: 0.1–0.2 nM, masitinib: 0.2–0.5 nM, nilotinib: 0.2–2 nM, dasatinib: 0.5–2 nM, sunitinib: 1–2 nM, and midostaurin: 5–10 nM. These drugs were also found to block the activity of PDGFR-downstream signaling molecules, including Akt, S6, and STAT5 in EOL-1 cells. Targeting of individual downstream molecules with specific inhibitors (PI3-kinase: NVP-BEZ235; mTOR: everolimus; STAT5: pimozide and piceatannol) also induced growth-inhibition in EOL-1 cells, although IC50 values were higher compared to that obtained with PDGFR-blocking TKI. All effective TKI produced dose-dependent apoptosis in EOL-1 cells as determined by microscopy, Annexin-V/PI staining, and staining for active caspase-3. In a next step, we applied the most effective TKI on primary neoplastic eosinophils. In these experiments, ponatinib, dasatinib, and nilotinib were found to suppress the growth of primary neoplastic eosinophils obtained from a patient with F/P+ CEL and one with ASM-eo, in a dose-dependent manner (IC50 <0.5 μM). In the patient with reactive HE, the TKI also produced growth inhibition, but IC50 values were higher compared to neoplastic eosinophils. We also examined drug effects on growth of Ba/F3 cells expressing the imatinib-resistant F/P mutants T674I and D842V. In these experiments, sunitinib was found to inhibit the growth of Ba/F3 cells expressing the T674I mutant of F/P. By contrast, no substantial effects of masitinib or nilotinib on Ba/F3 cells expressing this mutant were found, and Ba/F3 cells expressing F/P D842V were found to be resistant against sunitinib and masitinib. Strong inhibitory effects on both mutants were only seen with ponatinib. We next examined the effects of various TKI on cytokine-induced migration of neoplastic eosinophils. Unexpectedly, of all cytokines tested including IL-5 and eotaxin, only SDF-1A was found to induce in vitro migration of EOL-1 cells. We found that imatinib, nilotinib, dasatinib, ponatinib, sorafenib, and masitinib inhibit SDF-1A-induced migration of EOL-1 cells in a dose-dependent manner (effective range: 10–100 nM). Finally, we analyzed TKI effects on expression of activation-linked cell surface antigens on EOL-1 cells. In these experiments, we found that ponatinib and sorafenib downregulate expression of CD25 and CD63 in EOL-1 cells, whereas the other TKI tested showed no effects. By contrast, no effects of ponatinib or sorafenib on expression of HLA-DR, CXCR4 and CD95 on EOL-1 cells were seen. We were also unable to detect any significant effects of the other TKI on expression of activation-linked cell surface antigens in EOL-1 cells. In summary, our data show that various novel TKI counteract growth, survival, activation, and migration of neoplastic human eosinophils. The most potent agent that also blocks all known mutant-forms of F/P appears to be ponatinib. Novel PDGFR-targeting TKI, such as ponatinib, may be attractive alternative drugs for the treatment of imatinib-resistant or intolerant CEL. Disclosures:Valent:Phadia: Research Funding.

Interpreting Mammalian Target of Rapamycin and Cell Growth Inhibition in a Genetically Engineered Mouse Model of Nf1-Deficient Astrocytes

The identification of mammalian target of rapamycin (mTOR) as a major mediator of neurofibromatosis-1 (NF1) tumor growth has led to the initiation of clinical trials using rapamycin analogs. Previous studies from our laboratory have shown that durable responses to rapamycin treatment in a genetically engineered mouse model of Nf1 optic glioma require 20 mg/kg/day, whereas only transient tumor growth suppression was observed with 5 mg/kg/day rapamycin despite complete silencing of ribosomal S6 activity. To gain clinically relevant insights into the mechanism underlying this dose-dependent effect, we used Nf1-deficient glial cells in vitro and in vivo. First, there was an exponential relationship between blood and brain rapamycin levels. Second, we show that currently used biomarkers of mTOR pathway inhibition (phospho-S6, phospho-4EBP1, phospho-STAT3, and Jagged-1 levels) and tumor proliferation (Ki67) do not accurately reflect mTOR target inhibition or Nf1-deficient glial growth suppression. Third, the incomplete suppression of Nf1-deficient glial cell proliferation in vivo following 5 mg/kg/day rapamycin treatment reflects mTOR-mediated AKT activation, such that combined 5 mg/kg/day rapamycin and PI3-kinase (PI3K) inhibition or dual PI3K/mTOR inhibition recapitulates the growth suppressive effects of 20 mg/kg/day rapamycin. These new findings argue for the identification of more accurate biomarkers for rapamycin treatment response and provide reference preclinical data for comparing human rapamycin levels with target effects in the brain.

A Phase I Study of ENMD-2076 In Patients with Relapsed or Refractory Leukemia

AbstractAbstract 3307 Background:ENMD-2076 is a novel, orally-active molecule that inhibits Aurora A kinase and FLT3, as well as multiple receptor tyrosine kinases that drive tumor vascularization, including VEGFR2 (KDR), PDGFR and FGFR. A phase I study was conducted to determine the maximum tolerated dose (MTD) and toxicities of ENMD-2076 in patients with refractory hematological malignancies. Methods.Patients (cohorts of 6 evaluable patients per dose level) received escalating doses of ENMD-2076 administered orally daily [225 mg (n=7), 375 mg (n=6), 325 mg (n=9), or 275 mg (n=5)]. Plasma levels of ENMD-2076 were measured on Days 1, 8, & 29 of cycle 1 and at the end of study. Peripheral blood and/or bone marrow were obtained at baseline for ex vivo drug sensitivity testing, and on Days 8 & 29 of cycle 1 for pharmacodynamic (PD) monitoring to assess the effects of ENMD-2076 on cell signaling pathways. The latter flow cytometry-based assay used combined pERK, pAkt, pSTAT5, and pS6 labelling, and tested the effects of acute stimulation with SCF and FLT3 ligands in the presence or absence of pathway inhibitors, including ENMD-2076. Results.Twenty-seven patients have been treated to date (26 AML; 1 CMML-2). Median age is 69 years (range, 43–84 years). Median ECOG status 1 (range, 0 to 2). Eighteen patients (89%) had received prior therapy (median, 2 regimens; range, 0 to 6 regimens); 2 had received a prior allogeneic stem cell transplant. A total of 42 cycles have been administered, with a median of 1 cycle (range, 0 to 8 cycles); 10 patients (37%) have received 2 or more cycles of therapy. The most common nonhematological toxicities of any grade, regardless of association with drug, were fatigue, diarrhea, dysphonia, dyspnea, hypertension, constipation, and abdominal pain. Two of 6 patients treated at the 375 mg/day dose level developed dose-limiting toxicities (DLT), consisting of grade 3 fatigue. The dose was therefore decreased to 325 mg/day; one patient on this dose level developed a DLT with grade 3 fatigue. However, as 2 additional patients subsequently developed grade 2 or 3 fatigue (during cycles 1 and 2, respectively), the 325 mg/day dose was not believed to be tolerable for chronic administration in this patient population. The dose was therefore decreased to 275 mg/day; 2 patients subsequently developed DLTs consisting of grade 3 typhilitis and grade 3 syncope, respectively. Overall, no patient experienced grade 4 toxicities or death from ENMD-2076. Of the 20 evaluable patients, one patient achieved a CRi (transfusion-independent with platelets <100 × 109/L), three a morphologic leukemia-free state (MLFS) with a major HI-P and/or HI-E, and 4 other patients had an 11%, 14%, 23%, and 65% reduction in marrow blast count, respectively. Ex vivo studies showed that pre-incubation with ENMD-2076 in concentrations in the range of 0.5–2 mM suppressed growth factor stimulation in the blast cells of most patients tested to date. Decreases in the stimulation of ERK, Akt, STAT5, and S6 were seen in the Day 8 & 29 samples, including a striking inhibition of cell signaling in one patient who achieved a CRi. Conclusions.Single agent ENMD-2076 has activity in a heavily pretreated group of AML patients that may correlate with inhibition of ERK, Akt, STAT5, and S6 activity. The recommended phase two dose (RPTD) is 225 mg/day. Enrollment, as well as PK and PD monitoring of this study, is ongoing. Disclosures:Off Label Use: Clinical trial investigating ENMD-2076 in patients with relapsed or refractory leukemia. Sidor:EntreMed, Inc.: Employment. Fletcher:EntreMed, Inc.: Employment. Arnott:EntreMed, Inc.: Employment. Bray:EntreMed, Inc.: Employment.

Abstract A106: A phase 1 study of ENMD-2076 in patients with relapsed or refractory acute myeloid leukemia (AML)

Abstract ENMD-2076 is a novel, orally-active molecule that inhibits Aurora A kinase as well as multiple receptor tyrosine kinases that drive tumor vascularization, including VEGFR2 (KDR), PDGFR and FGFR. A phase 1 study was conducted to determine the maximum tolerated dose (MTD) and toxicities of ENMD-2076 in patients with refractory hematological malignancies. Fifteen patients with AML (cohorts of 6 patients per dose level) have been treated with 225 (n=7), 325 (n=2), and 375 (n=6) mg of ENMD-2076 administered orally once daily. Median age was 76 years (range, 60 to 82 years). Median ECOG status was 1 (range, 0 to 2). Fourteen patients had received prior therapy (median, 2 regimens; range, 0 to 4 regimens). A total of 17 cycles have been administered to date, with a median of 1 cycle (range, 0 to 3 cycles); 2 patients (14%) have received 3 or more cycles of therapy. The most common ENMD-2076-related adverse events were grade ≤ 2 and consisted of dizziness, petechiae, hypertension, nausea, fatigue, diarrhea, and reflux. Dose-limiting toxicity consisted of grade 3 fatigue in 2 patients at the 375 mg/day dose level. Therefore, the dose was decreased to 325 mg/day. Following a drug holiday, both patients who had experienced the grade 3 fatigue were restarted at the 325 mg/day dose level. No patient experienced grade 4 toxicities or death from ENMD-2076. Of the 13 evaluable patients, 1 patient achieved a morphologic leukemic free state (MLFS) with platelet transfusion independence. One patient achieved a HI-P. Two other patients had a 12% and 14% reduction in marrow blast count, respectively. At the time of analysis, 3 patients discontinued therapy due to disease progression. Peripheral blood and/or bone marrow were obtained at baseline for ex vivo drug sensitivity testing, and on Days 8 and 29 of cycle 1 for pharmacodynamic (PD) monitoring, using a whole blood flow cytometry protocol to measure ENMD-2076 effects on cell signalling pathways. This assay uses combined labelling for P-ERK, P-Akt, P-STAT5, and P-S6 as the readout, and tests the effects of acute stimulation with the ligands SCF and FL in the presence or absence of pathway inhibitors, including ENMD-2076. Pre-incubation with drug concentrations in the range 0.5 – 2 µM suppressed growth factor stimulation in the blast cells of all patients tested to date. Decreases in the ability to stimulate ERK,Akt, STAT5, and S6 were seen in the Day 8 and 29 samples, including a striking inhibition of cell signalling in one patient who achieved a MLFS. Assays are also in progress to monitor specific effects on Aurora kinase and the cell cycle in these patient samples. In conclusion, single agent ENMD-2076 has activity in a heavily pretreated group of AML patients that may correlate with inhibition of ERK,Akt, STAT5, and S6 activity. Enrollment, as well as PK and PD monitoring of this study, is ongoing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A106.

The effect of the protein level in a pre-starter diet on the post-hatch performance and activation of ribosomal protein S6 kinase in muscle of neonatal broilers

The cytoplasmic serine/threonine ribosomal protein S6 kinase (S6K1) plays a critical role in controlling protein translation. There is evidence that amino acids regulate S6K1 and protein synthesis in avian species, but the effect of dietary protein level on the activation of S6K1 in neonatal chicks is unknown. Therefore, the aim of the present experiment was to investigate the effect of different protein levels, supplied during the first 5 d post-hatch, on body growth, breast muscle development and on the activation of S6K1 and its downstream target, the S6, in neonatal chicks. Chicks were fed a pre-starter diet during the first 5 d post-hatch containing low (19.6 % crude protein (CP); LP), medium (23.1 % CP; MP) or high (26.7 % CP) levels (HP) of protein. Weight gain of chicks fed the HP diet was higher (P < 0.05) compared with those fed the LP diet during day (d)3-d5 and the numerical advantage of this group was maintained from d2 to d7. On d2 and d3, greater levels of S6K1 and S6 phosphorylation and/or activity were observed in chicks receiving the HP diet compared with LP and MP diets, without differences between results of the latter two dietary treatments. In conclusion, the present results suggest that early protein nutrition impacts the development of broiler chicks.

Pharmacodynamic (PD) study of pre-prostatectomy rapamycin in men with advanced localized prostate cancer (PC): A DOD Prostate Cancer Clinical Trials Consortium Trial

5001 Background: Rapamycin is an orally bioavailable and generic mTOR inhibitor with an MTD of 6 mg daily and anticancer activity in solid tumors. We sought to determine the optimal biologic dose (OBD) in the pre-operative setting in men with PC. Methods: We explored the safety and PD activity of 3 and 6 mg of daily oral rapamycin for 14 days prior to radical prostatectomy (RP) in cohorts of 21 men with intermediate risk localized PC. Ten untreated control subjects were included using identical inclusion criteria. Men had Gleason &gt;6 PC involving multiple cores. PD markers in pre-treatment prostate biopsies and RP specimens included p-S6 and p-Akt, 4EBP-1, PTEN, p27, Ki-67, and cleaved caspase-3. Tissue and whole blood sirolimus levels were measured. A Simon 2-stage design using PD efficacy (tumor S6 inhibition &gt; 60%) was utilized. Results: 32 subjects were accrued to this 2 stage pharmacodynamic trial, including 10 control subjects. Median pre-treatment PSA was 6.4 ng/dl, age 60y, Gleason 7 in 85%. 20 accrued to the 3 mg cohort without DLT. However, 2/2 men enrolled at the 6 mg dose level experienced DLT consisting of thrombocytopenia leading to a delay in surgery (&lt;100K) and fever with grade 3 stomatitis. AEs observed in the 3 mg cohort included stomatitis (2), rash (1), post-operative ileus (2), and mild neutropenia (2). One post-operative ileus was observed among control patients. PD studies demonstrated tumor S6 inhibition in &gt;50% of subjects (median 60% decline, p=0.026 vs. baseline) with no negative effect on the state of Akt phosphorylation (p=0.82) or p27 levels (p=0.10). Prostate sirolimus levels (range 7.1–47.2 ng/g) were two-fold higher than whole blood concentrations (range 3.2–19.2 ng/ml). There was no effect of rapamycin on rates of post-operative wound healing or bleeding. PBMC S6 activity inhibition did not correlate with tumor S6 inhibition. Conclusions: The MTD/OBD of daily rapamycin in the pre-operative or prevention setting is 3 mg. This dose demonstrated downstream mTOR inhibition in tumor tissue and achieved adequate prostate tissue levels. Additional PD and genomic studies will be presented. Conducted through the DOD PCCTC and registered at clinicaltrials.gov as NCT00311623 . [Table: see text]

Cyclosporine A from a nonpathogenic Fusarium oxysporum suppressing Sclerotinia sclerotiorum

To evaluate the antagonistic activity of Fusarium oxysporum nonpathogenic fungal strain S6 against the phytopathogenic fungus Sclerotinia sclerotiorum and to identify the antifungal compounds involved. The antagonistic activity of Fusarium oxysporum strain S6 was determined in vitro by dual cultures. The metabolite responsible for the activity was isolated by chromatographic techniques, purified and identified by spectroscopic methods as cyclosporine A. The antifungal activity against the pathogen was correlated with the presence of this metabolite by a dilution assay and then quantified. Cyclosporine A caused both growth inhibition and suppression of sclerotia formation. In a greenhouse assay, a significant increase in the number of surviving soybean (Glycine max) plants was observed when S. sclerotiorum and F. oxysporum (S6) were inoculated together when compared with plants inoculated with S. sclerotiorum alone. Fusarium oxysporum (S6) may be a good fungal biological control agent for S. sclerotiorum and cyclosporine A is the responsible metabolite involved in its antagonistic activity in vitro. Cyclosporine A has not been previously described as an inhibitor of S. sclerotiorum. Its minimum inhibitory concentration (MIC) of 0.1 microg disc(-1) makes it suitable to use as a biofungicide. In vivo experiments showed that F. oxysporum (S6) is a good candidate for the biocontrol of S. sclerotiorum in soybean.

E3 ubiquitin ligase RNF2 interacts with the S6′ proteasomal ATPase subunit and increases the ATP hydrolysis activity of S6′

We reported previously that the human RNF2 (RING finger protein 2) protein is an E3 ubiquitin ligase that interacts with the human ubiquitin-conjugating enzyme Hip-2/hE2-25K. In the present study, we show that RNF2 interacts with S6' ATPase, a subunit of the proteasomal 19 S regulatory complex. S6' interacts with RNF2 through its N-terminal RING domain, and RNF2 interacts with S6' through its C-terminal region. Interestingly, the RNF2-S6' interaction increases the ATP hydrolysis activity of the S6' protein. Moreover, S6' ATPase activity is highly increased in the presence of ubiquitinated proteins. The present study suggests that the E3 ubiquitin ligase RNF2 might have a dual function: facilitating the ubiquitination of its target substrates and recruiting the substrates to the proteasome. Furthermore, ATP hydrolysis in the E3/proteasome complex might act as an important signal for the protein degradation pathway.

Tuberin Regulates p70 S6 Kinase Activation and Ribosomal Protein S6 Phosphorylation: A ROLE FOR THE TSC2 TUMOR SUPPRESSOR GENE IN PULMONARY LYMPHANGIOLEIOMYOMATOSIS (LAM)

Although the cellular functions of TSC2 and its protein product, tuberin, are not known, somatic mutations in the TSC2 tumor suppressor gene are associated with tumor development in lymphangioleiomyomatosis (LAM). We found that ribosomal protein S6 (S6), which exerts translational control of protein synthesis and is required for cell growth, is hyperphosphorylated in the smooth muscle-like cell lesions of LAM patients compared with smooth muscle cells from normal human blood vessels and trachea. Smooth muscle (SM) cells derived from these lesions (LAMD-SM) also exhibited S6 hyperphosphorylation, constitutive activation of p70 S6 kinase (p70S6K), and increased basal DNA synthesis. In parallel, TSC2-/- smooth muscle cells (ELT3) and TSC2-/- epithelial cells (ERC15) also exhibited hyperphosphorylation of S6, constitutive activation of p70S6K, and increased basal DNA synthesis. Re-introduction of wild type tuberin into LAMD-SM, ELT3, and ERC15 cells abolished phosphorylation of S6 and significantly inhibited p70S6K activity and DNA synthesis. Rapamycin, an immunosuppressant, inhibited hyperphosphorylation of S6, p70S6K activation, and DNA synthesis in LAMD-SM cells. Interestingly, the basal levels of phosphatidylinositol 3-kinase, Akt/protein kinase B, and p42/p44 MAPK activation were unchanged in LAMD-SM and ELT3 cells relative to levels in normal human tracheal and vascular SM. These data demonstrate that tuberin negatively regulates the activity of S6 and p70S6K specifically, and suggest a potential mechanism for abnormal cell growth in LAM.

Effects of ML-9 on insulin stimulation of glucose transport in 3T3-L1 adipocytes.

Treatment of 3T3-L1 adipocytes with insulin resulted in activation of 2-deoxyglucose transport activity and translocation of glucose transporters (GLUT4 and GLUT1) from the cytoplasmic space to the plasma membrane. ML-9 (a myosin light chain kinase inhibitor) inhibited insulin stimulation of 2-deoxyglucose transport activity by 80% at 100 microM (IC50 = 27 microM) without affecting 2-deoxyglucose transport activity in the basal state. The inhibition was independent of extracellular Ca2+ concentration and almost fully reversible at 40 microM ML-9. ML-9 did not inhibit insulin-stimulated tyrosine phosphorylation of 95-kDa protein in the wheat germ agglutinin-purified preparation and of 95- and 160-kDa proteins in intact cells. However, ML-9 inhibited insulin-induced translocation of both GLUT4 and GLUT1 in a dose-dependent manner. The dose-response curves were similar to those observed for the inhibition of insulin stimulation of 2-deoxyglucose transport activity. Neither insulin nor ML-9 affected the phosphorylation state of both heavy and light chains of myosin. Therefore, it seems likely that ML-9 inhibits the insulin-induced translocation of glucose transporters at a step beyond the insulin receptor kinase activity by a mechanism different from that affecting phosphorylation of the myosin light chain. Phosphorylating activity of microtubule-associated protein 2 and myelin basic protein was stimulated by insulin, and this stimulation was not affected by ML-9. ML-9, however, inhibited the phosphorylating activity in vitro and insulin stimulation of the phosphorylating activity of ribosomal protein S6 in intact cells in a dose-dependent manner similar to that observed for the inhibition of insulin stimulation of glucose transport. These results suggest that mitogen-activated protein kinase may be one of the constituents in intracellular insulin signaling to the glucose transport system.

Inhibition of Staphylococcal Enterotoxin B Formation by Cell Wall Blocking Agents and other Compounds

Enterotoxin B formation by Staphylococcus aureus S6 was inhibited by Tween 80, oleic acid, sodium deoxycholate, penicillin, d-cycloserine, or bacitracin. Toxin formation by strain 243 was sensitive to oleic acid, sodium deoxycholate, sodium lauryl sulfate, d-cycloserine, or bacitracin. The effect of d-cycloserine was reversed by d-alanine with strain 243 but not with strain S6. Neither penicillin nor bacitracin inhibited alpha-hemolysin or coagulase activity of strain S6; however, 0.118 mumoles of d-cycloserine per ml increased the alpha-hemolysin titer more than eightfold. Pigmentation of strain 243 was reduced by oleic acid, sodium deoxycholate, or methicillin, and was completely inhibited by d-cycloserine or bacitracin. Glucose was required for the inhibition by spermine of (14)C-valine incorporation into cellular protein of strain S6. These data indicate that the cell surface may contain sites important to the synthesis of enterotoxin B.