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J W Simons

Publications and source records attributed to J W Simons.

At least 37 records · Page 2Linked to original sources
PubMed1999

Direct comparison of GAPDH, beta-actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia.

The appropriate choice of an internal standard is critical for quantitative RNA analyses. As housekeeping genes, GAPDH, beta-actin, cyclophilin, and 28S rRNA are commonly employed as RNA internal standards with the assumption that their expression levels remain relatively constant in different experimental conditions. We tested this assumption under hypoxia (1% O2, 24 hours) compared to normoxia (20% O2, 24 hours) and compared RNA levels of these 4 housekeeping genes head to head using ribonuclease protection assays. Four biologically diverse cell lines with respect to clonal origin, neoplastic transformation, and growth rates were used in the comparison. Expression levels of 28S rRNA were constant, independent of O2 tension, but levels of GAPDH, beta-actin, and cyclophilin varied widely with hypoxia. In particular, GAPDH mRNA expression was increased by 21.2-75.1% under hypoxic conditions. Increased GAPDH transcription in hypoxia was correlated in the cancer cell lines with upregulation of the transcription factor Hypoxia Inducible Factor-1alpha protein levels in identical experimental conditions. These results suggest that 28S rRNA is a reliable internal control for comparative analyses of transcription under hypoxia; GAPDH appears particularly unfavorable for this purpose either in hypoxia or other experimental conditions that upregulate HIF-1alpha.

Actins↗
PubMed1999

Rational design of alpha-keto triglyceride analogues as inhibitors for Staphylococcus hyicus lipase.

We have synthesized a series of alpha-keto triglyceride analogues as inhibitors for the lipase from Staphylococcus hyicus (SHL). Hydrolysis at positions 1 and 2 was prevented by replacement of the ester bonds by nonhydrolyzable ether, carbamoyl, or amide bonds, and an alpha-keto fatty acid was introduced at position 3. Such compounds act as competitive inhibitors of SHL. Inhibition must be caused by the presence of the alpha-keto functions, since the compounds containing an ester or a hydroxyl group in position 3 did not inhibit the enzyme. We propose that our inhibitors react with the active site Ser of the lipase, hereby mimicking the tetrahedral intermediate occurring in substrate hydrolysis. We conclude that the localization of the alpha-keto triglycerides is very important for inhibition because only those compounds which are insoluble in water are lipase inhibitors. In addition, other specific protein-inhibitor interactions, probably with the carbonyl oxygen at position 1 and/or 2, improve inhibitor binding. This makes the compounds with amide or carbamoyl groups at positions 1 and 2 better inhibitors than their ether counterparts. The inhibitory power could be improved further by replacing the oxygen at position 3 by an amido group. The resulting inhibitor 1, 2-diethylcarbamoyl-3-amido-alpha-ketododecanoylglycerol has a Ki value of 0.008 mol %, indicating that it binds approximately 125-fold tighter than the substrate. These results illustrate that effective lipase inhibitors can be designed by combining an alpha-keto group with good micellar solubility and optimal protein-inhibitor interactions.

Drug Design↗
PubMed1999

Characterization of the role of IL-6 in the progression of prostate cancer.

BACKGROUND: We recently identified IL-6, a pleiotropic cytokine implicated in the neoplastic process of a variety of neoplasms, as a mediator of prostate cancer morbidity. In the present study, we investigated the expression of members of the IL-6 supergene family and related cytokines and the potential role of IL-6 in prostate cancer growth regulation. METHODS: Five established human prostate cancer cell lines were screened by ELISA for production of granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), tumor necrosis factor (TNF), interleukin-1 (IL-1), and granulocyte macrophage colony-stimulating factor (GM-CSF). Expression of the ligand-binding component of the IL-6 receptor, IL-6Rp80, was evaluated by ELISA and RT-PCR. Sequential immunoprecipitation and immunoblotting were performed to assay for expression of the signal-transducing component of the IL-6 receptor, gp130. The effects of IL-6 on cell growth were assessed by MTT assays. RESULTS: The three hormone-refractory cell lines, DU-145, TSU, and PC-3, secreted distinct combinations of cytokines (DU-145: IL-6, GM-CSF; TSU: IL-6, LIF; PC-3: IL-6, G-CSF, LIF, IL-1, GM-CSF), each uniformly expressing IL-6. In contrast, neither of the two hormone-dependent cell lines, LNCaP-ATCC and LNCaP-GW, secreted significant quantities of any of the cytokines analyzed. None of the cell lines secreted detectable quantities of OSM, CNTF, or TNF. All cell lines, irrespective of hormone status, expressed both Il-6Rp80 and gp130. Addition of IL-6 in vitro inhibited growth of hormone-dependent cells, but had no effect on hormone-refractory lines. Anti-IL-6 neutralizing antibody inhibited growth of hormone-refractory cells. CONCLUSIONS: IL-6 appears to undergo a functional transition from paracrine growth inhibitor to autocrine growth stimulator during progression of prostate cancer to the hormone-refractory phenotype.

Cell Division↗
PubMed1999

Identification of a calcium binding site in Staphylococcus hyicus lipase: generation of calcium-independent variants.

In this study we have identified the presence of a high-affinity binding site for calcium in the lipase from Staphylococcus hyicus. By means of isothermal titration calorimetry we showed that the enzyme binds one calcium per molecule of enzyme with a dissociation constant of 55 microM. The residual activity of the apoenzyme compared to the activity in the presence of calcium ions varies from 65% at 10 degreesC to nearly zero at 40 degreesC. On the basis of primary sequence alignment with other staphylococcal lipases and the lipases from Bacillus thermocatenulatus and from Pseudomonas glumae in combination with site-directed mutagenesis, aspartates 354 and 357 could be identified as calcium ligands. Kinetic measurements with the D357E variant showed that replacement of Asp357 by a glutamate decreased the affinity for calcium ions 30-fold. Introduction of a lysine, an asparagine, or an alanine at position 357 and of a lysine or an asparagine at position 354 resulted in calcium-independent variants. Isothermal titration calorimetry confirmed the loss of calcium binding. Although the D357K, D357N, and D357A variants did not bind calcium, at room temperature they were nearly as active as wild-type lipase in the presence of calcium, but at elevated temperatures these calcium-independent lipases showed a reduced activity. Over the whole temperature range the activities of the D354K and D354N variants are significantly lower than wild-type enzyme in the presence of calcium and are comparable to the activity of the wild-type apoenzyme. Our results show that binding of calcium is important for the structural stabilization of staphylococcal lipases (and possibly other lipases) and that it is possible to engineer calcium-independent variants on the basis of limited structural homology with another lipase.

Amino Acid Sequence↗
PubMed1999

Nuclear morphometry adds significant prognostic information to stage and grade for renal cell carcinoma.

OBJECTIVES: Identification of patients with a high probability of recurrence after nephrectomy for renal cell carcinoma (RCC) is required for adjuvant studies of new therapies. Nuclear morphometry predicts prognosis for prostate, bladder, and Wilms' tumors and in RCC according to previous small pilot studies. METHODS: To validate this finding, we studied an additional 101 patients who underwent nephrectomy for Stage pT1 to pT3 RCC at our institution from 1977 to 1993 for whom data regarding recurrence or disease-free survival of greater than 60 months were available. Patient records and pathology specimens were reviewed. Of the 101 patients, 66 (65%) did not experience recurrence with greater than 60 months of follow-up, and 35 (35%) had RCC recurrence with a median time to recurrence of 17 months. Nuclear shape descriptors were tested as predictors of disease recurrence after accounting for stage and grade in proportional hazards regression models. RESULTS: Range of ellipticity (hazards ratio 3.39, P = 0.014) was confirmed to be a significant predictor of recurrence. A prognostic model using stage, grade, and range of ellipticity identified three distinct groups: low, moderate, and high recurrence risk groups, with recurrence rates of 4%, 37%, and 63%, respectively, at 5 years of follow-up. Morphometry significantly (P = 0.018) improved prognostication on the basis of stage and grade alone in this multivariate model. CONCLUSIONS: Nuclear morphometry is valid and accurate in predicting relapse in early-stage RCC. The model can select patients with RCC for adjuvant therapies.

Carcinoma, Renal Cell↗
PubMed1998

Increased expression of hypoxia inducible factor-1alpha in rat and human prostate cancer.

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates genes involved in adaptation to hypoxia. Expression of HIF-1alpha was evaluated in rat and human prostate cancer cell lines. Increased expression of HIF-1alpha mRNA in rat prostate cancer cell lines and hypoxia-induced expression of HIF-1alpha protein in human prostate cancer cell lines are associated with increased cell growth rates and metastatic potential. HIF-1alpha mRNA was undetectable in the normal rat ventral prostate by Northern blot hybridization. HIF-1alpha protein expression and HIF-1 DNA binding activity were detected in normoxic PC-3 cells. Human prostate cancer cells plated at low density manifested higher functional HIF-1alpha expression than cells plated at high density independent of O2 tension. HIF-1alpha may become dysregulated in prostate cancer and thus drive the transcription of hypoxia-adaptive genes involved in tumor progression. This is also the first evidence that human cancer cells can express functional HIF-1alpha protein under normoxic conditions.

Animals↗
PubMed1998

Impaired expression and function of signal-transducing zeta chains in peripheral T cells and natural killer cells in patients with prostate cancer.

Detection of functional, circulating T cells and NK cells may serve as a clinical test for the selection of individuals who can benefit from immunotherapy. Incidence of the T-cell receptor zeta (TCRdelta) chain within these populations appears to correlate with adequate effector cell function. In patients with advanced malignancy, the absence or reduced expression of delta chain has been documented. Flow cytometric analysis in the present study revealed a significant reduction in delta chain expression in peripheral blood lymphocytes (PBL) of 14 of 22 prostate cancer patients (P < 0.000001) as compared to normal donors, apparent in both T cells (CD3+, CD4+, CD8+), and NK (CD16+) cells. Compared to normal donor PBL, patient PBL cultured in the presence of CD3 and CD28, also demonstrated reduced expression of CD69 and/or CD25, and in some cases, failed to activate at all. Furthermore, evidence of cell proliferation in activation-stimulated patient PBL was muted: average PCNA positivity equaled 14%, a marked difference from what was observed in normal donors (P < 0.0002). In 8 of 16 samples of PBL, where delta expression was originally low, delta levels returned to the normal range after 48 hour culture in serum-free medium, suggesting that the loss of delta is reversible and may be caused by a tumor-derived substance. These data support the premise that monitoring the expression of delta in a cancer patient may provide a unique insight into the immune status and functionality of the individual, with the potential to redirect or augment therapies and ultimately alter prognosis.

Adult↗
PubMed1998

Cloning, purification and characterisation of the lipase from Staphylococcus epidermidis--comparison of the substrate selectivity with those of other microbial lipases.

On the chromosome of Staphylococcus epidermidis RP62A the lipase gene (gehSE1) is immediately flanked by the icaAA'BC operon, which is involved in biofilm formation. Since lipase production might play a role in staphylococcal skin colonisation as well, we studied the biochemical properties of the staphylococcal lipases more closely. The DNA sequence and the deduced protein sequence revealed that gehSE1 is very similar to the lipase sequence of S. epidermidis strain 9. Like other staphylococcal lipases, gehSE1 is organised as a preproenzyme. The part of gehSE1 coding for the mature lipase was cloned and overexpressed as a fusion protein with an N-terminal histidine tag in Escherichia coli. The lipase was purified to homogeneity using a combination of precipitation techniques, metal-affinity chromatography and gel filtration. Biochemical characterisation showed that this lipase is closely related to the lipase from Staphylococcus aurelis NCTC8530. Both enzymes have a pH optimum around 6, are very stable at low pH, and need calcium as a cofactor for catalytic activity. The preferred substrates are small triacylglycerols, with a maximum activity toward tributyrylglycerol. Comparison of the substrate selectivity with those of other microbial lipases showed that phospholipids are generally poor substrates for lipases. An exception is the lipase from Staphylococcus hyicus, which prefers phospholipids as a substrate, distinguishing this staphylococcal lipase from other microbial lipases. These results are discussed in view of the structure/function relationships of staphylococcal lipases, and the possible involvement of these enzymes in biological processes such as skin colonisation and pathogenesis.

Amino Acid Sequence↗
PubMed1998

Identification of diphtheria toxin via screening as a potent cell cycle and p53-independent cytotoxin for human prostate cancer therapeutics.

BACKGROUND: Metastatic human prostate cancer requires novel therapeutic strategies in order to overcome its low proliferative rate and its resistance to conventional chemotherapeutic agents. To identify potential cytotoxin gene products for use in experimental therapeutics such as in vivo gene therapy, an in vitro screen was designed. METHODS: Eight recombinant cellular toxins were tested for activity against a spectrum of metastatic human prostate cancer cell phenotypes. Pseudomonas exotoxin A, ricin, tumor necrosis factor alpha (TNF-alpha), diphtheria toxin (DT), Crotalus durissus terrificus toxin, crotalus adamenteus toxin, Naja naja toxin, and Naja mocambique toxin were evaluated. Comparative survival distinguished the relative potencies of these cytotoxins for irreparable prostate cancer cell death. RESULTS: Of the phospholipase A2 toxins, Crotalus durissus terrificus and Naja mocambique are active against the PSA secreting LNCaP cell line; however, the effect is reversible, and no other hormone refractory prostate cell line tested is sensitive. Screening identified toxin-specific differences: dose-dependent cytotoxic activity against all human prostate cancer cell lines tested was only identified for ricin and diphtheria toxin (DT) as highly potent. DT has an IC50 in the range of 20-00 pM by clonogenic survival and kills irreversibly by both apoptosis as well as nonapototic pathways. Acquisition of p53 mutant status conferred no reduction in sensitivity to DT cytotoxicity. Cell cycle arrest by aphidicolin did not protect human prostate cells from irreversible DT-induced cell death. TNF-alpha had modest cytostatic activity in the screen; however, the combination of TNF-alpha and DT resulted in marked acceleration of the time to prostate cancer cell death. CONCLUSIONS: The rational screening of cytotoxins allows the identification of cell cycle-independent agents of variable potency against human prostate cancer. DT-mediated cell death is cell cycle independent, and p53 independent, making it particularly attractive for application to cytoreductive gene therapy, targeted monoclonal antibodies, and prodrug delivery of toxins applied to human prostate cancer therapeutics.

Antineoplastic Agents↗
PubMed1998

Biochemical properties of staphylococcal (phospho)lipases.

Various staphylococci secrete lipases which require calcium ions for activity, and have profound preferences for substrates with different chain lengths. The lipase from Staphylococcus hyicus is exceptional since it has higher phospholipase than lipase activity. This paper gives an overview of the biochemical properties of these enzymes. It appears that chain length selectivity of these enzymes resides in the acylation step. Interfaces mainly influence the acylation step. Calcium ions do not influence the rate of acylation or deacylation although stabilise the enzyme against denaturation. Molecular modelling based on the X-ray structure of Pseudomonas glumae lipase was used to construct a model of the staphylococcal lipases. With this model the position of serveral residues involved in stubstrate selectivity was predicted. Moreover, a sequence element could be assigned that may function as the so-called lid domain in staphylococcal lipases. Sequence alignment of four staphylococcal lipases, and lipases from P. glumae and Bacillus thermocatenulatus identified several potential calcium ligands, one of which was verified by site directed mutagensesis. It is concluded that stabilisation of lipases by calcium ions might be a more general phenomenon than recognized so far.

Amino Acid Sequence↗
PubMed1998

The phospholipase activity of Staphylococcus hyicus lipase strongly depends on a single Ser to Val mutation.

Site-directed mutagenesis and domain exchange were used to investigate the role of the C-terminal domains of Staphylococcus hyicus lipase (SHL) and S. aureus lipase (SAL) in substrate selectivity. The introduction of a single point mutation coding for the substitution of Val for Ser356 in SHL yields an enzyme which has retained full lipase activity, but with more than 12-fold lower phospholipase activity. Starting with this S356V variant of SHL the C-terminal 40 amino acids were replaced by the corresponding SAL sequence. Although 23 amino acid changes were introduced simultaneously the impact on the phospholipase/lipase activity ratio was only 4-fold. We therefore conclude that in the C-terminal domain it is Ser356 which mainly determines phospholipase activity. The introduction of a Val357 to Ser substitution in SAL did not turn SAL into a phospholipase, showing that residues from other domains contribute to this activity as well. The results are discussed in view of the sequence homology of lipases and (lyso)phospholipases.

Amino Acid Sequence↗
PubMed1998

Serum vascular endothelial growth factor is a candidate biomarker of metastatic tumor response to ex vivo gene therapy of renal cell cancer.

We report the close correlation between changes in serum immunoreactive vascular endothelial growth factor 165 (iVEGF165) levels and metastatic tumor burden measured by computed tomography scan before treatment, during the antitumor response, and during early progression in a patient treated with ex vivo gene therapy for renal cell carcinoma. With the researcher blinded to outcome, iVEGF levels were measured in archived serum samples from a patient with metastatic renal cell carcinoma who demonstrated a 7-month partial remission to treatment with autologous, irradiated human GM-CSF gene transduced tumor vaccine. Although a spontaneous regression could not be formally excluded in this patient, the appearance of 20 new pulmonary metastases on computed tomography scan after nephrectomy and before vaccination indicates that if spontaneous regression occurred, it took place at the start of vaccine treatment.

Biomarkers, Tumor↗
PubMed1998

The future of gene therapy in the treatment of urologic malignancies.

Gene therapy as a field of academic inquiry and as a medical technology is perhaps the inevitable consequence of living in today's technologic age. At its essence, gene therapy is merely therapy using molecular information. The identification of the pivotal role of DNA in the information systems of life and the subsequent emergence of molecular biology and its technologies has led to routine use of the genetic code-like software to treat human disease in clinical trials.

Forecasting↗
PubMed1998

Cytotoxic effects of sodium phenylbutyrate on human neuroblastoma cell lines.

Sodium phenylbutyrate (NaPB) is used in urea cycle disorders. We screened 6 neuroblastoma cell lines for in vitro potency of NaPB as an antiproliferative agent, evaluated multiple dosing schedules, and assessed its activity in combination with clinically active agents for neuroblastoma. We determined that NaPB achieves a 30-80% growth inhibition at 5 mM. Repeated dosing and prolonged drug exposure enhanced the cytotoxic effect. NaPB had additive cytotoxic effects when administered with vincristine; however, NaPB did not affect the activity of etoposide, adriamycin, 4-hydroxycyclophosphamide or cisplatinum. These results suggest that NaPB is an active agent against neuroblastoma and could be combined with vincristine in novel chemotherapy regimens.

Antineoplastic Agents↗
PubMed1998

Ex-vivo gene therapy using cytokine-transduced tumor vaccines: molecular and clinical pharmacology.

Whether the current generation of cytokine gene-transduced tumor vaccines will show clinical efficacy is under study. Fortunately, the large safety profile so far observed with gene-transduced tumor vaccines can allow outpatient testing in large populations of patients in the adjuvant therapy situation. This will allow large studies statistically powered to see potentially important adjuvant therapy effects in the range that are observed for tamoxifen in breast cancer. For example, the outpatient, adjuvant therapy safety context has been established in the use of GM-CSF gene-transduced autologous prostate cancer vaccines following radical prostatectomy. Similar adjuvant therapy clinical trial efforts are anticipated with allogeneic breast, colon, pancreatic, and ovarian cancer in addition to prostate, renal cell carcinoma, and melanoma. The reverse translation of early clinical data back to basic laboratory research also suggests the field of cytokine gene-transduced tumor vaccine research will remain vibrant. Efforts are currently being directed on optimizing DC activation with polycistronic constructs of cytokine genes, and overexpressing the most relevant tumor-associated peptides. As in the case of antineoplastic drug development, not all lead compounds will become approved drugs in medical oncology. Rigorous yet innovative clinical trial designs will be key to the accelerated identification of cytokine gene-transduced vaccines that improve survival in cancer patients.

Animals↗
PubMed1997

Dissecting the catalytic mechanism of staphylococcal lipases using carbamate substrates: chain length selectivity, interfacial activation, and cofactor dependence.

p-Nitrophenyl N-alkylcarbamates with different alkyl chains were used as substrates to determine separately the carbamylation and decarbamylation rates of the lipases from Staphylococcus hyicus and S. aureus. Both enzymes are reversibly inhibited by these compounds due to a rapid carbamylation of their active site serines followed by a slow decarbamylation. The carbamylation reaction is strongly pH-dependent and the pH profile suggests that an unprotonated histidine is required for this reaction. In contrast, the decarbamylation is pH-independent suggesting the presence of a hydrogen bond between the active site histidine and the carbamyl moiety. S. hyicus lipase preferably reacts with medium to long chain carbamates with an optimum for eight carbon atoms. In contrast, S. aureus lipase is highly specific for short chain carbamates. These results are in agreement with the respective substrate preferences of both lipases toward natural lipids. The decarbamylation rates of both enzymes hardly depend on the alkyl chain length, and from this it is concluded that chain length selectivity is expressed in the first step of catalysis. Both the carbamylation and decarbamylation reaction rates of S. hyicus lipase are enhanced in the presence of micelles, the activation effect being most pronounced in the first step. For the S. aureus lipase only a small influence of interfaces on both reaction steps was observed. These results are discussed in view of a possible role of a lid covering the active site. Kinetic experiments in the presence and absence of calcium strongly suggest that calcium ions are important for the structural stabilization of the unmodified as well as of the carbamylated enzymes. This structural function of calcium was supported by urea unfolding experiments, from which it appeared that for both enzymes the free energy for unfolding is significantly lower in the absence of calcium. In conclusion our results show that kinetic differences between both lipases reside in the acylation step, and that calcium is important for the structural stabilization of the unmodified, and moreover, the acylated enzymes.

Carbamates↗