ROLE OF GLUTATHIONE IN FORMALDEHYDE METABOLISM AND TOXICITY

Glutathione (GSH) is involved in the detoxication of numerous chemicals exogenously exposed or endogenously generated. Exposure to these agents cause depletion of cellular GSH rendering these cells more susceptible to the toxic action of these same agents. Formaldehyde (CH(,2)O) was found to deplete cellular GSH, presumably by the formation of the GSH-CH(,2)O complex, S-hydroxymethylglutathione, and its rapid extrusion into the extracellular medium.^ The metabolism and toxicity of CH(,2)O were determined to be dependent upon cellular GSH in vitro and in vivo. The rate of CH(,2)O oxidation decreased and the extent of toxicity increased when isolated rat hepatocytes or strain A/J mice were pretreated with the GSH-depleting agent, diethyl maleate (DEM). Additional experiments were designed to further study the role GSH plays in detoxication using isolated rat hepatocytes.^ L-Methionine protected against the extent of lipid peroxidation and leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), caused by CH(,2)O in DEM-pretreated hepatocytes, further supporting the protective role of GSH against cellular toxicity. The antioxidants, ascorbate, butylated hydroxytoluene, and (alpha)-tocopherol, were all protective against the extent of lipid peroxidation and leakage of LDH in isolated rat hepatocytes. Whereas L-methionine may be protective by increasing the cellular concentration of GSH which is used to detoxify free radicals or by facilitating the rate of CH(,2)O oxidation, the antioxidant, ascorbate, was protective without altering the rate of CH(,2)O oxidation or increasing cellular GSH levels. These results suggest that the free radical-mediated toxicity caused by CH(,2)O in DEM-pretreated hepatocytes is due to the further depletion of GSH by CH(,2)O and not to increased CH(,2)O persistence. How this further depletion in GSH by CH(,2)O in DEM-pretreated hepatocytes results in lipid peroxidation and cell death was further investigated.^ The further decrease in GSH caused by CH(,2)O in DEM-pretreated hepatocytes, suspected of stimulating lipid peroxidation and cell death, was found not to be due to depletion of mitochondrial GSH but to depletion of protein sulfhydryl groups. In addition, cellular toxicity appears more closely correlated with depletion of protein sulfhydryl groups than with an increase in cytosolic free Ca('2+). The combination of CH(,2)O and DEM may be a useful tool in identifying these critical sulfhydryl-protein(s) and to further understand the role GSH plays in detoxication. ^

Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs.

The injurious effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the small intestine was not appreciated until the widespread use of capsule endoscopy. Animal studies found that NSAID-induced small intestinal injury depends on the ability of these drugs to be secreted into the bile. Because the individual toxicity of amphiphilic bile acids and NSAIDs directly correlates with their interactions with phospholipid membranes, we propose that the presence of both NSAIDs and bile acids alters their individual physicochemical properties and enhances the disruptive effect on cell membranes and overall cytotoxicity. We utilized in vitro gastric AGS and intestinal IEC-6 cells and found that combinations of bile acid, deoxycholic acid (DC), taurodeoxycholic acid, glycodeoxycholic acid, and the NSAID indomethacin (Indo) significantly increased cell plasma membrane permeability and became more cytotoxic than these agents alone. We confirmed this finding by measuring liposome permeability and intramembrane packing in synthetic model membranes exposed to DC, Indo, or combinations of both agents. By measuring physicochemical parameters, such as fluorescence resonance energy transfer and membrane surface charge, we found that Indo associated with phosphatidylcholine and promoted the molecular aggregation of DC and potential formation of larger and isolated bile acid complexes within either biomembranes or bile acid-lipid mixed micelles, which leads to membrane disruption. In this study, we demonstrated increased cytotoxicity of combinations of bile acid and NSAID and provided a molecular mechanism for the observed toxicity. This mechanism potentially contributes to the NSAID-induced injury in the small bowel.

The inhibition of lipid transfer protein by negatively charged liposomes modifies the therapeutic index of amphotericin B

We have shown that liposomal amphotericin B (L-AmpB) decreased renal toxicity and maintains the antifungal activity of amphotericin B (AmpB). We have also observed that L-AmpB is predominantly associated with high density lipoproteins (HDL) as compared to Fungizone (AmpB + deoxycholate). The present experiments were designed to assess the biological relevance of transferring AmpB to HDL. We observed that AmpB was less toxic to kidney cells when associated with HDL, however AmpB toxicity was maintained when associated with LDL. To further understand how HDL-associated AmpB reduces renal cell toxicity the presence of HDL and LDL receptors in this cell line was determined. We observed that these cells expressed high and low affinity LDL receptors, but only low affinity HDL receptors. The reduced renal cell toxicity of HDL-associated AmpB may be due to its lack of interaction with renal cells because of the absence of HDL receptors. Since AmpB interacts with cholesteryl esters whose transfer among lipoproteins is regulated by Lipid transfer Protein (LTP), the role of LTP on the distribution of AmpB to HDL and LDL was next examined. We found that negatively charged liposomes significantly reduced LTP-mediated transfer of CE between HDL and LDL, independent of the presence of AmpB, while Fungizone only significantly inhibited CE transfer at one concentration tested (20$\mu$g/ml). Therefore, we believe that the decreased renal toxicity of L-AmpB is related to its predominant distribution to HDL which is regulated by the inhibition of LTP activity. ^

A PHARMACOLOGICAL EVALUATION OF THE MECHANISM OF CYTOTOXICITY OF 9-BETA-D- XYLOFURANOSYLADENINE IN CHINESE HAMSTER OVARY CELLS

The biochemical determinants of cytotoxicity of the purine nucleoside analog, 9-(beta)-D-xylofuranosyladenine (xyl-A) were studied in wild-type Chinese hamster ovary cells and in nucleoside kinase deficient mutants. It was found that {('3)H}xyl-A was readily phosphorylated to the triphosphate level in both the wild-type and deoxycytidine kinase deficient mutant, but not by the adenosine kinase deficient cells. Values for the apparent Km and Vmax of this uptake process were 43.9 (mu)M and 118.7 nmol/min/10('9) cells, respectively. Cloning procedures indicated that the viability of CHO cells was decreased 90 per cent by a 5-hr incubation with 10 (mu)M xyl-A. However, the toxicity of xyl-A was increased 100-fold by the addition of a nontoxic concentration (10 (mu)M) of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to the medium. High-pressure liquid chromatographic analysis indicated that after 5 hr, the concentration of 9-(beta)-D-xylofuranosyladenine 5'-triphosphate (xyl-ATP) in cells incubated with xyl-A plus EHNA was 2.0 mM, four times greater than in those cells incubated with xyl-A alone. Incubation with xyl-A plus EHNA had no significant effect on the cellular concentrations of 5-phosphoribosyl-1-pyrophosphate after 1 hr whereas, treatment with 3'-dexoyadenosine (cordycepin) decreased the concentration of this metabolite. Determinations of the cellular nucleoside triphosphates indicated that under conditions that resulted in an intracellular accumulation of 500 (mu)M xyl-ATP, the endogenous concentrations of neither the ribonucleoside triphosphates nor deoxyribonucleoside triphosphates were significantly different from those of control cells. The ID(,50) for {('3)H}thymidine incorporation into DNA, 105 (mu)M xyl-ATP, was four-fold less than the ID(,50) for {('3)H}uridine incorporation into RNA suggesting that the process of DNA synthesis is more sensitive to the presence of xyl-ATP. When removed from exogenous xyl-A, CHO cells failed to recover their ability to synthesize RNA and DNA, although the intracellular xyl-ATP concentration decreased to less than 35 (mu)M. The selective inhibition of RNA synthesis by 6-azauridine did not prevent the expression of toxicity by xyl-ATP. However, the selective inhibition of DNA synthesis by ara-C significantly spared toxicity in cells that had accumulated an otherwise lethal concentration of xyl-ATP. It is shown that in cells which had accumulated 1.27 mM {('3)H}xyl-ATP, {('3)H}xyl-A was found to terminate cellular RNA chains at a frequency of 1.42 (mu)mol of {('3)H}xyl-A 3' termini per mol of mononucleotide. These results indicate that a general mechanism for the toxicity of xyl-A to CHO cells includes the cellular accumulation of xyl-ATP, which serves as a substrate for RNA synthesizing enzymes and subsequently is incorporated into nascent RNA transcripts as a chain terminator. A specific mechanism involving the premature termination of RNA primers required for the initiation of DNA synthesis is proposed to account for the inhibitory action of xyl-ATP on DNA synthesis. ^

THE ESTROGENIC PROPERTIES OF O,P'-DDT

o,p'-DDT is a major component of the pesticide DDT (dichlorodiphenyltrichloro ethane, technical grade). Although possessing little insecticidal ability, the o,p'- isomer has two major biological activities which affect mammalian reproductive systems: it is estrogenic, and it induces hepatic mixed function oxidase enzymes. The focus of this work is the characterization of the estrogenic properties of o,p'-DDT in rodents.^ Initial studies examined the ability of o,p'-DDT to bind to and interact with elements of the estrogen receptor system. In an in vitro assay, DDT was shown to compete with 17(beta)-estradiol (E(,2)) for binding to cytoplasmic estrogen receptors (R(,c)) from normal and neoplastic tissues in two rodent species. The following phenomena were studied by measuring receptor levels from uteri (whole uteri and/or uterine cell types) taken from immature ovariectomized rats given one acute injection of o,p'-DDT or E(,2): the translocation of the R(,c) to the nucleus, nuclear receptor (R(,n)) retention patterns, and the subsequent reappearance of R(,c) in the cytoplasm.^ The magnitude and temporal patterns of the biological responses of uteri from similar immature rats were compared following o,p'-DDT and E(,2) exposure. The responses examined included increased "Induced Protein" synthesis (in vitro); and uterine wet weight, DNA synthesis and mitosis (in vivo).^ From dose-response data, correlations were made between R(,n) levels and levels of subsequent biological responses. The aim was to lend support to the premise that biological responses to o,p'-DDT exposure occur as a result of its interaction with the classical estrogen receptor system. Correlation coefficients of 0.95 to 0.98 were obtained between R(,n) levels and levels of responses examined, strongly supporting this hypothesis.^ Finally, o,p'-DDT was shown to be as effective as E(,2) in supporting the growth of a transplantable estrogen-responsive mammary tumor in adult rats (although it was unable to support the growth of a transplantable estrogen-dependent renal tumor in hamsters). While the positive result cannot be directly extrapolated to human or animal exposure to environmental estrogens, it suggests that hyperplastic responses of estrogen sensitive tissues should be considered as a possible toxicity of o,p'-DDT, related compounds having estrogenic properties, and other environmental estrogens. ^

Lindane induced cellular dysfunction in MDCK cells: The role of the peripheral benzodiazepine receptor

The central nervous system GABAA/Benzodiazepine (GABAA/BZD) receptors are targets for many pharmaceutical agents and several classes of pesticides. Lindane is an organochlorine pesticide, although banned from production in the U.S. since 1977, still imported for use as an insecticide and pharmaceutically to control ectoparasites (ATSDR, 1994). Lindane functions as a GABA/BZD receptor antagonist within the central nervous system (CNS). Outside of the CNS, peripheral BZD receptors have been localized to the distal tubule of the kidney. Previous research in our laboratory has shown that incubation of renal cortical slices with lindane can produce an increase in kallikrein leakage, suggesting a distal tubular effect. In this study, Madin Darby Canine Kidney (MDCK) cells were used as an in vitro system to assess the toxicity of lindane. This purpose of this study was to determine if interactions between a renal distal tubular BZD-like receptor and lindane could lead to perturbations in renal distal cellular chloride (Cl−) transport and mitochondrial dysfunction and ultimately, cellular death. ^ Pertubations in renal chloride transport were measured indirectly by determining if lindane altered cell function responsiveness following osmotic stress. MDCK cells pre-treated with lindane and then subjected to osmotic stress remained swollen for up to 12 hours post-stress. Lindane-induced dysfunction was assessed through stress protein induction measured by Western Blot analysis. Lindane pretreatment delayed Heat Shock Protein 72 (HSP72) induction by 36 hours in osmotically stressed cells. Pretreatment with 1 × 10 −5 M LIN followed by osmotic stress elevated p38 and Stress Activated Protein Kinase (SAPK/JNK) at 15 minutes which declined at 30 minutes. Lindane appeared to have no effect on Endoplasmic Reticulum Related Kinase (ERK) induction. Lindane did not effect osmotically stressed LLC-PKI cells, a control cell line. ^ Lindane-treated MDCK cells did not exhibit necrosis. Instead, apoptosis was observed in lindane-treated MDCK cells in both time- and dose-dependent manners. LLC-PKI cells were not affected by LIN treatment. ^ To better understand the mechanism of lindane-induced apoptosis, mitochondrial function was measured. No changes in cytochrome c release or mitochondrial membrane potential were observed suggesting the mitochondrial pathway was not involved in lindane-induced apoptosis. ^ Further research will need to be conducted to determine the mechanism of lindane-induced adverse cellular effects. ^

Importance of sperm transition proteins demonstrated in mice carrying Tnp1- and Tnp2-null alleles

Chromatin condensation within the nucleus of developing spermatids involves replacement of histones by transition proteins, which are in turn replaced by protamines. The importance of transition proteins in the complex process of spermiogenesis has, to date, been only speculative. This study sought to investigate the extent to which transition proteins are essential or have redundant functions by characterizing sperm produced in mice expressing all combinations of Tnp-null alleles. Results from breeding trials of 8 weeks duration revealed that, on average, wildtype males produced about 14 offspring whereas TP2 and TP1 single-knockout males produced about 8 and 1 offspring, respectively, demonstrating their subfertility. Genotypes with less than two Tnp wildtype alleles, as well as double-knockout mutants, were completely infertile. Sperm from males with impaired fertility had poor progressive motility, heterogeneous chromatin condensation, incompletely processed protamine 2 and head and tail abnormalities. Generally, as the number of Tnp-null alleles increased so did the severity of abnormalities. However, specific morphological abnormalities were associated with the absence of an individual TP. Studies which sought to identify possible root causes for abnormalities in thiol-rich sperm structures revealed no differences in thiol content or sulfhydryl oxidation status within the nucleus but nuclei and tails from single-knockout mutants were severely disrupted following thiol reduction. Binding of fluorescent dyes to DNA was normal in sperm recovered from caput but abnormal in cauda epididymal sperm from TP1 knockouts and infertile double mutants. Injection of cauda epididymal sperm from double knockouts into oocytes produced very few offspring; however, after injection with testicular sperm, the efficiency was no different from wildtype. These results suggest DNA structural alterations or degradation during epididymal transport of sperm resulting in a diminished capacity of the paternal DNA of these sperm to produce offspring. The overall importance of transition proteins for normal chromatin condensation and production of fertile sperm has been demonstrated. Furthermore, identification of specific morphological abnormalities associated with the absence of an individual transition protein provides new evidence that the proteins are not completely redundant and each fulfills some unique function. ^

Preclinical modeling of multi-drug cancer therapies

Anticancer drugs typically are administered in the clinic in the form of mixtures, sometimes called combinations. Only in rare cases, however, are mixtures approved as drugs. Rather, research on mixtures tends to occur after single drugs have been approved. The goal of this research project was to develop modeling approaches that would encourage rational preclinical mixture design. To this end, a series of models were developed. First, several QSAR classification models were constructed to predict the cytotoxicity, oral clearance, and acute systemic toxicity of drugs. The QSAR models were applied to a set of over 115,000 natural compounds in order to identify promising ones for testing in mixtures. Second, an improved method was developed to assess synergistic, antagonistic, and additive effects between drugs in a mixture. This method, dubbed the MixLow method, is similar to the Median-Effect method, the de facto standard for assessing drug interactions. The primary difference between the two is that the MixLow method uses a nonlinear mixed-effects model to estimate parameters of concentration-effect curves, rather than an ordinary least squares procedure. Parameter estimators produced by the MixLow method were more precise than those produced by the Median-Effect Method, and coverage of Loewe index confidence intervals was superior. Third, a model was developed to predict drug interactions based on scores obtained from virtual docking experiments. This represents a novel approach for modeling drug mixtures and was more useful for the data modeled here than competing approaches. The model was applied to cytotoxicity data for 45 mixtures, each composed of up to 10 selected drugs. One drug, doxorubicin, was a standard chemotherapy agent and the others were well-known natural compounds including curcumin, EGCG, quercetin, and rhein. Predictions of synergism/antagonism were made for all possible fixed-ratio mixtures, cytotoxicities of the 10 best-scoring mixtures were tested, and drug interactions were assessed. Predicted and observed responses were highly correlated (r2 = 0.83). Results suggested that some mixtures allowed up to an 11-fold reduction of doxorubicin concentrations without sacrificing efficacy. Taken together, the models developed in this project present a general approach to rational design of mixtures during preclinical drug development. ^

Homeostatic transcriptional regulation of bcl-2 in the epidermis

Bcl-2, a crucial regulator of cell survival, is frequently overexpressed in basal cell carcinomas (BCCs), the most commonly diagnosed cancers. Regulation of bcl-2 expression in epidermal keratinocytes is not well characterized. In the epidermis, bcl-2 is expressed only in keratinocytes of the basal layer and the outer root sheath of hair follicles and no bcl-2 expression in suprabasalar keratinocytes. The calcium gradient in the epidermis is a potent regulator of keratinocyte differentiation. Increasing calcium concentrations associated with differentiation, resulted in the downregulation of a 2.9 kb bcl-2 promoter luciferase construct. The AP-1 family of transcription factors is differentially expressed in the strata of the epidermis and has been shown to be involved in the stage specific expression of numerous differentiation markers in the epidermis. In silico analysis of the bcl-2 promoter and gene reporter assays showed that co-transfection of JUNB and JUND, but not other AP-1 dimers, caused a significant upregulation of the bcl-2 promoter in primary keratinocytes. Immunoelectrophoretic mobility shift assays, in vivo chromatin immunoprecipitation (ChIP) studies and mutational analysis of AP-1 binding site 3 on the bcl-2 promoter identified it as the site involved in bcl-2 regulation. Utilizing site directed mutants, we determined that phosphorylation at Ser90/Ser100 residues of JUND is required for the activation of the bcl-2 promoter. ^ The sonic hedgehog (SHH) pathway is frequently deregulated in BCCs and, we have shown that GLI1 upregulates bcl-2 in keratinocytes. While examining potential regulation of the SHH pathway extracellular calcium, we found that higher calcium concentrations are associated with lowered HH pathway activity and upregulation of suppressor of fused (SUFU) which negatively regulates the SHH pathway. ChIP assays, and in vivo mouse models, show that ΔNp63α, a crucial regulator of epidermal development, binds and activates the SUFU promoter in differentiating keratinocytes. Increasing SUFU levels prevent transactivation of the bcl-2 promoter. In vitro SUFU knockdown along with in vivo SUFU+/− murine models demonstrate a significant upregulation of bcl-2 expression. ^ In conclusion, the spatial and temporal expression of bcl-2 during keratinocyte differentiation in the epidermis is a complex process requiring cooperative interactions of specific signaling cascades and transcription factors. ^

DEVELOPMENT AND APPLICATION OF SHORT-TERM MUTAGENICITY AND CYTOTOXICITY BIOASSAYS FOR INTEGRATED HEALTH ASSESSMENT STUDIES OF COAL FLY ASH EMISSIONS

Two respirable coal fly ash samples ((LESSTHEQ) 3(mu)m), one from a pressurized fluidized-bed combustion miniplant and one from a conventional combustion power plant, were investigated for physical properties, chemical composition and biological activity. Electron microscopy illustrated irregularity in fluidized-bed combustion fly ash and sphericity in conventional combustion fly ash. Elemental analysis of these samples showed differences in trace elements. Both fly ash samples were toxic in rabbit alveolar macrophage and Chinese hamster ovary cell systems in vitro. The macrophages were more sensitive to toxicity of fly ash than the ovary cells. For measuring the cytotoxicity of fly ash, the most sensitive parameters were adenosine triphosphate in the alveolar macrophage system and viability index in the hamster ovary system. Intact fluidized-bed combustion fly-ash particles showed mutagenicity only in strains TA98 and TA1538 without metabolic activation in the Ames Salmonella assay. No mutagenicity was detected in bioassay of conventional combustion fly ash particles. Solvent extraction yielded more mass from fluidized-bed combustion fly ash than from conventional combustion fly ash. The extracts of fluidized-bed combustion fly ash showed higher mutagenic activity than conventional combustion fly ash. These samples contained direct-acting, frameshift mutagens.^ Fly ash samples collected from the same fluidized-bed source by cyclones, a fabric filter, and a electrostatic precipitator at various temperatures were compared for particle size, toxicity, and mutagenicity. Results demonstrated that the biological activity of coal fly ash were affected by the collection site, device, and temperature.^ Coal fly ash vapor-coated with 1-nitropyrene was developed as a model system to study the bioavailability and recovery of nitroaromatic compounds in fly ash. The effects of vapor deposition on toxicity and mutagenicity of fly ash were examined. The nitropyrene coating did not significantly alter the ash's cytotoxicity. Nitropyrene was bioavailable in the biological media, and a significant percentage was not recovered after the coated fly ash was cultured with alveolar macrophages. 1-Nitropyrene loss increased as the number of macrophages was increased, suggesting that the macrophages are capable of metabolizing or binding 1-nitropyrene present in coal fly ash. ^

Assessing high-priority mixtures: A review of methanol as influenced by ethanol

The intent of this research was to identify the level of risk methanol posed to a fetus during an ethanol co-exposure. This investigation was prompted by the known competitive inhibition properties of ethanol and the developmental toxicity of methanol. Integrated into this research was the practicality necessitated by regulatory processes, namely: does the risk justify the expense of additional research. To this end, the scope and nature of exposures were summarized to illustrate the ubiquity of these chemicals and the potential for dual exposure. Similarly, severity of outcome was evaluated by systematically reviewing the LOAELs, NOAELs, and statistical significance contained in methanol-induced developmental studies. Results. Blood methanol levels corresponding to developmental effects in laboratory studies were found to be substantially higher than the blood methanol levels predicted in high-risk methanol-ethanol exposure scenarios. This indicates that ethanol would not likely exacerbate methanol toxicity to the point of teratogenicity; however, it is important to note that the developmental toxicity of ethanol—an established human teratogen—was not included in the evaluation. Ethanol's contribution as a developmental toxicant rather than merely as an attenuator of methanol toxicity undermines the severity of effects possible from this chemical combination. Therefore further evaluation is needed to assess the developmental toxicities following dual exposures before rendering methanol and ethanol a high-priority mixture.^

INTERACTION OF BACILLUS ANTHRACIS EXOSPORIUM PROTEIN BCLA WITH COMPLEMENT FACTOR H AND SPORE PERSISTENCE IN THE LUNG

Anthrax outbreaks in the United States and Europe and its potential use as a bioweapon have made Bacillus anthracis an interest of study. Anthrax infections are caused by the entry of B. anthracis spores into the host via the respiratory system, the gastrointestinal tract, cuts or wounds in the skin, and injection. Among these four forms, inhalational anthrax has the highest lethality rate and persistence of spores in the lungs of animals following pulmonary exposure has been noted for decades. However, details or mechanisms of spore persistence were not known. In this study, we investigated spore persistence in a mouse model. The results suggest that B. anthracis spores have special properties that promote persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence. Moreover, recent discoveries from our laboratory suggest that spores evolved a sophisticated mechanism to interact with the host complement system. The complement system is a crucial part of the host defense mechanism against foreign microorganisms. Knowledge of the specific interactions that occur between the complement system and B. anthracis was limited. Studies performed in our laboratory have suggested that spores of B. anthracis can target specific proteins, such as Factor H (fH) of the complement system. Spores of B. anthracis are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called Bacillus collagen-like protein of anthracis (BclA), which comprises a central collagen-like region and a globular C-terminal domain. BclA is the first point of contact with the innate system of an infected host. In this study, we investigated the molecular details of BclA-fH interaction with respect to the specific binding mechanism and the functional significance of this interaction in a murine model of anthrax infection. We hypothesized that the recruitment of fH to the spore surface by BclA limits the extent of complement activation and promotes pathogen survival and persistence in the infected host. Findings from this study are significant to understanding how to treat post-exposure prophylaxis and improve our knowledge of spores with the host immune system.

A simulation study of the standard design, the rolling six design, the CRM, and the modified CRM in Phase I clinical trials

The Phase I clinical trial is considered the "first in human" study in medical research to examine the toxicity of a new agent. It determines the maximum tolerable dose (MTD) of a new agent, i.e., the highest dose in which toxicity is still acceptable. Several phase I clinical trial designs have been proposed in the past 30 years. The well known standard method, so called the 3+3 design, is widely accepted by clinicians since it is the easiest to implement and it does not need a statistical calculation. Continual reassessment method (CRM), a design uses Bayesian method, has been rising in popularity in the last two decades. Several variants of the CRM design have also been suggested in numerous statistical literatures. Rolling six is a new method introduced in pediatric oncology in 2008, which claims to shorten the trial duration as compared to the 3+3 design. The goal of the present research was to simulate clinical trials and compare these phase I clinical trial designs. Patient population was created by discrete event simulation (DES) method. The characteristics of the patients were generated by several distributions with the parameters derived from a historical phase I clinical trial data review. Patients were then selected and enrolled in clinical trials, each of which uses the 3+3 design, the rolling six, or the CRM design. Five scenarios of dose-toxicity relationship were used to compare the performance of the phase I clinical trial designs. One thousand trials were simulated per phase I clinical trial design per dose-toxicity scenario. The results showed the rolling six design was not superior to the 3+3 design in terms of trial duration. The time to trial completion was comparable between the rolling six and the 3+3 design. However, they both shorten the duration as compared to the two CRM designs. Both CRMs were superior to the 3+3 design and the rolling six in accuracy of MTD estimation. The 3+3 design and rolling six tended to assign more patients to undesired lower dose levels. The toxicities were slightly greater in the CRMs.^

Characterization of Jak, STAT, and Src interactions in Head and Neck Squamous Cell Carcinoma

Recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is common; thus, it is essential to improve the effectiveness and reduce toxicity of current treatments. Proteins in the Src/Jak/STAT pathway represent potential therapeutic targets, as this pathway is hyperactive in HNSCC and it has roles in cell migration, metastasis, proliferation, survival, and angiogenesis. During short-term Src inhibition, Janus kinase (Jak) 2, and signal transducer and activator of transcription (STAT) 3 and STAT5 are dephosphorylated and inactivated. Following sustained Src inhibition, STAT5 remains inactive, but Jak2 and STAT3 are reactivated following their early inhibition. To further characterize the mechanism of this novel feedback pathway we performed several experiments to look at the interactions between Src, Jak2, STAT5 and STAT3. We attempted to develop a non-radioactive kinase assay using purified recombinant Jak2 and Src proteins, but found that phospho-tyrosine antibodies were non-specifically binding to purified recombinant proteins. We then performed in vitro kinase assays (IVKAs) using purified recombinant Jak2, Src, STAT3, and STAT5 proteins with and without Src and Jak2 pharmacologic inhibitors. We also examined the interactions of these proteins in intact HNSCC cells. We found that recombinant Jak2, STAT3, and STAT5 are direct substrates of Src and that recombinant Src, STAT3, and STAT5 are direct substrates of Jak2 in the IVKA. To our knowledge, the finding that Src is a Jak substrate is novel and has not been shown before. In intact HNSCC cells we find that STAT3 can be reactivated despite continuous Src inhibition and that STAT5 continues to be inhibited despite Jak2 reactivation. Also, Jak2 inhibition did not affect Src or STAT5 activity but it did cause STAT3 inhibition. We hypothesized that the differences between the intact cells and the IVKA assays were due to a potential need for binding partners in intact HNSCC cells. One potential binding partner that we examined is the epidermal growth factor receptor (EGFR). We found that EGFR activation caused increased activation of Src and STAT5 but not Jak2. Our results demonstrate that although STAT3 and STAT5 are capable of being Src and Jak2 substrates, in intact HNSCC cells Src predominantly regulates STAT5 and Jak2 regulates STAT3. Regulation of STAT5 by Src may involve interactions between Src and EGFR. This knowledge along with future studies will better define the mechanisms of STAT regulation in HNSCC cells and ultimately result in an ideal combination of therapeutic agents for HNSCC.