Drug Sensitivity Studies Research Articles

Asynchronous magnetic bead rotation microviscometer for rapid, sensitive, and label-free studies of bacterial growth and drug sensitivity.

The long turnaround time in antimicrobial susceptibility testing (AST) endangers patients and encourages the administration of wide spectrum antibiotics, thus resulting in alarming increases of multidrug resistant pathogens. A method for faster detection of bacterial proliferation presents one avenue toward addressing this global concern. We report on a label-free asynchronous magnetic bead rotation (AMBR) based viscometry method that rapidly detects bacterial growth and determines drug sensitivity by measuring changes in the suspension's viscosity. With this platform, we observed the growth of a uropathogenic Escherichia coli isolate, with an initial concentration of 50 cells per drop, within 20 min; in addition, we determined the gentamicin minimum inhibitory concentration (MIC) of the E. coli isolate within 100 min. We thus demonstrated a label-free, microviscometer platform that can measure bacterial growth and drug susceptibility more rapidly, with lower initial bacterial counts than existing commercial systems, and potentially with any microbial strains.

Abstract B125: Identification and characterization of a panel of B-Raf wildtype and mutant patient-derived low passage tumor models.

Abstract Activating mutations of the BRAF gene occurs in approximately 60% of melanomas, most often resulting in a V600E mutant. Other substitutions at this and adjacent residues are known to exist, resulting in constitutive B-raf kinase activity and poor clinical prognosis. B-raf mutations may also play a role in the pathogenesis of a number of other malignancies including colorectal, lung, thyroid and hematopoietic cancers. Due to the importance of this target in cancer progression, several potential therapies targeting B-raf are currently in preclinical and clinical development. To better understand the role of B-raf in melanoma and other cancers we have established and characterized a panel of wildtype and mutated B-raf melanoma, colorectal, ovary and biliary cancer patient-derived tumor models. For each model, tissue was implanted into immune-deficient mice and once established, each model was confirmed by histologic analysis and linked with donor patient treatment and outcome data. In addition, model characterization including mutation profiling and drug sensitivity studies were performed and results compared with clinical information. Finally we evaluated relevant B-raf wildtype and mutant models with the B-raf inhibitor vemurafenib. Eighteen low passage melanoma models have been established, nine with confirmed mutated BRAF genes. Four additional mutant B-Raf tumor models include two colorectal one ovary and one biliary cancer. Model information and identified mutations are as follows: Additional gene mutations identified include KRAS (9%), NRAS (9%), TP53 (14%) and CTNNB1 (5%). Differential activity was reported for single agent temozolomide, carboplatin and paclitaxel in melanoma model studies. In the majority, there was no correlation between mutation status and cytotoxic drug sensitivity. However, two B-raf models with identical mutations demonstrated sensitivity to irinotecan with sustained partial tumor regressions. Additional drug screens of two multi-mutated models, ST004 and ST054, showed resistance to all evaluated therapies. The ST274 (BRAF-D594G) melanoma model was found insensitive to vemurafenib; results from other models are pending. Conclusion: We have generated and a panel of patient-derived tumor models which include B-raf wildtype and mutated melanoma, colorectal, ovary and biliary cancers possessing differential mutation status and drug sensitivity profiles. Expansion of this panel of tumors is planned and should further enhance its utility in development of useful agents towards melanoma and other B-raf mutant cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B125.

Abstract B17: Establishment, characterization and evaluation of a panel of patient-derived colorectal tumor models.

Abstract Background: Patient-derived tumor models passaged only a few times in vivo retain physical and molecular characteristics of human cancer and may prove essential in identifying disease biomarkers and drug targets in later stage development. Currently we have collected tumor tissue from chemo-naïve and pretreated patients and established and characterized a panel of low passage colorectal tumor models. Methods: For each model, tissue was implanted into immune-deficient mice and once established, each model was confirmed by histologic analysis and linked with donor patient treatment and outcome data. Model characterization including mutation profiling and drug sensitivity studies were performed and results compared with clinical patient information; each model was evaluated with the EGFR inhibitor cetuximab and two standard clinical therapies FOLFOX and irinotecan and compared with patient's drug sensitivity before and after tissue sample collection. Single agent tumor growth inhibition was evaluated and study endpoints included mean control tumor volumes of approximately 1 cm3 or up to sixty days following treatment initiation. Results: Eighteen low passage colorectal models have been established and characterized. KRAS mutations were identified in 44% of models and activating mutations identified in some models include: PI3KCA (6%), TP53 (33%) and BRAF (6%). In vivo response to FOLFOX and irinotecan correlated to ≥65% of donor patient outcome to these treatments. Cetuximab sensitivity did not fully correlate with Ras mutation status as 4/6 wildtype and 4/8 Ras demonstrated cetuximab sensitivity in these studies. Conclusion: We have generated a panel of low passage colorectal tumor models with differential mutation status and drug sensitivity profiles. Expansion of this panel of patient-derived tumors is planned and should further enhance its utility in identifying agents potentially useful for treatment of colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B17.

Abstract PL03-01: Discovery of candidate biomarkers of anticancer drug sensitivity by high-throughput cell line screening.

Abstract PL03-01: Discovery of candidate biomarkers of anticancer drug sensitivity by high-throughput cell line screening.

Abstract B13: Identifying new potential therapies in ovarian cancer: Establishment and characterization of a panel of low passage tumor models and in vivo evaluation of approved and investigational agents.

Abstract Background: Ovarian cancer is the leading cause of death from gynecologic malignancies in Western countries. Standard treatment for ovarian cancer consists of tumor debulking followed by platinum/taxane therapy. While the rate of initial response to treatment is high, the majority of patients develop recurrent, chemoresistant disease. Secondary chemotherapy treatment provides benefit to only 10–30% of these patients, demonstrating the need for identifying additional useful therapies. To assist in identifying new therapies for ovarian cancer, we collected tumor tissue from chemo-naïve and pretreated patients and established, developed and characterized a panel of low passage ovarian tumor models. We then utilized these models to demonstrate antitumor activity of a novel targeted therapy. Methods: For each model, tissue was collected and implanted into immune-deficient mice and progress monitored. Each established model was confirmed by histologic comparison to the corresponding patient clinical sample and linked to their available treatment and outcome data. Additional characterization, including mutation profiling and standard drug sensitivity studies, was performed on each model and results compared with clinical information. An investigator-initiated study was performed evaluating activity of the Smac (second mitochondrial-derived activator of caspases) mimetic TL32711 in a subset of these models. Single agent tumor growth inhibition and delay were evaluated and study endpoints included mean and individual tumor volumes of approximately 1 cm3 or up to sixty days following treatment initiation. Results: Twenty low passage ovarian models have been established and characterized. Activating mutations identified in some models include: PI3KCA (6%), TP53 (19%), FBXW7 (6%), RAS (13%) and BRAF (6%). In vivo response to platinum and/or taxane agents correlated ≥70% with donor patient outcome at the time of sample collection and models demonstrated variable sensitivity to evaluated secondary chemotherapies. Single agent TL32711 showed statistically significant (p<0.05) treatment-dependent tumor growth inhibition and notable tumor growth delay in evaluated models. Conclusion: We have generated a panel of characterized patient-derived low passage ovarian tumor models with differential mutation status and drug sensitivity profiles. Using these models we have identified a preclinically active agent towards ovarian cancer. Expansion of this panel of ovarian tumors is planned and should further enhance its utility in identifying agents potentially useful for treatment of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B13.

Molecular targeted approaches for treatment of pancreatic cancer.

Human pancreatic cancer remains a highly malignant disease with almost similar incidence and mortality despite extensive research. Many targeted therapies are under development. However, clinical investigation showed that single targeted therapies and most combined therapies were not able to improve the prognosis of this disease, even though some of these therapies had excellent anti-tumor effects in pre-clinical models. Cross-talk between cell proliferation signaling pathways may be an important phenomenon in pancreatic cancer, which may result in cancer cell survival even though some pathways are blocked by targeted therapy. Pancreatic cancer may possess different characteristics and targets in different stages of pathogenesis, maintenance and metastasis. Sensitivity to therapy may also vary for cancer cells at different stages. The unique pancreatic cancer structure with abundant stroma creates a tumor microenvironment with hypoxia and low blood perfusion rate, which prevents drug delivery to cancer cells. In this review, the most commonly investigated targeted therapies in pancreatic cancer treatment are discussed. However, how to combine these targeted therapies and/or combine them with chemotherapy to improve the survival rate of pancreatic cancer is still a challenge. Genomic and proteomic studies using pancreatic cancer samples obtained from either biopsy or surgery are recommended to individualize tumor characters and to perform drug sensitivity study in order to design a tailored therapy with minimal side effects. These studies may help to further investigate tumor pathogenesis, maintenance and metastasis to create cellular expression profiles at different stages. Integration of the information obtained needs to be performed from multiple levels and dimensions in order to develop a successful targeted therapy.

Resistance of a Rodent Malaria Parasite to a Thymidylate Synthase Inhibitor Induces an Apoptotic Parasite Death and Imposes a Huge Cost of Fitness

BackgroundThe greatest impediment to effective malaria control is drug resistance in Plasmodium falciparum, and thus understanding how resistance impacts on the parasite's fitness and pathogenicity may aid in malaria control strategy.Methodology/Principal FindingsTo generate resistance, P. berghei NK65 was subjected to 5-fluoroorotate (FOA, an inhibitor of thymidylate synthase, TS) pressure in mice. After 15 generations of drug pressure, the 2% DT (the delay time for proliferation of parasites to 2% parasitaemia, relative to untreated wild-type controls) reduced from 8 days to 4, equalling the controls. Drug sensitivity studies confirmed that FOA-resistance was stable. During serial passaging in the absence of drug, resistant parasite maintained low growth rates (parasitaemia, 15.5%±2.9, 7 dpi) relative to the wild-type (45.6%±8.4), translating into resistance cost of fitness of 66.0%. The resistant parasite showed an apoptosis-like death, as confirmed by light and transmission electron microscopy and corroborated by oligonucleosomal DNA fragmentation.Conclusions/SignificanceThe resistant parasite was less fit than the wild-type, which implies that in the absence of drug pressure in the field, the wild-type alleles may expand and allow drugs withdrawn due to resistance to be reintroduced. FOA resistance led to depleted dTTP pools, causing thymineless parasite death via apoptosis. This supports the tenet that unicellular eukaryotes, like metazoans, also undergo apoptosis. This is the first report where resistance to a chemical stimulus and not the stimulus itself is shown to induce apoptosis in a unicellular parasite. This finding is relevant in cancer therapy, since thymineless cell death induced by resistance to TS-inhibitors can further be optimized via inhibition of pyrimidine salvage enzymes, thus providing a synergistic impact. We conclude that since apoptosis is a process that can be pharmacologically modulated, the parasite's apoptotic machinery may be exploited as a novel drug target in malaria and other protozoan diseases of medical importance.

Establishment of a Primary Pleomorphic Xanthoastrocytoma Cell Line

Anaplastic pleomorphic xanthoastrocytoma is an aggressively growing, malignant, and eventually fatal tumor of the central nervous system. Testing chemotherapeutic drug sensitivity under in vitro conditions would be a useful strategy to determine sensitive or resistant drugs for fatal brain cancers. To establish primary cell cultures of excised tumor tissue from pleomorphic xanthoastrocytoma-bearing patients and to test their sensitivity against various anticancer chemotherapy drugs. Prepared suspensions of the excised tumor tissue from a patient who had a recurrent grade 3 pleomorphic xanthoastrocytoma was cultured in culture dishes until cells began to grow. Immunofluorescent and immunohistochemical visualizations were performed using confocal and light microscopy. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in comparison with ³H-thymidine incorporation assay was used to test cellular toxicity of several anticancer drugs. We established vigorously growing primary cells of the tumor. Drug sensitivity testing was conducted successfully. Primary cell cultures of surgically removed tumor tissues may be useful in studies of cancer biology and chemotherapeutic drug sensitivity for recurrent malignant brain tumors, particularly for anaplastic pleomorphic xanthoastrocytoma.

Abstract 4426: Preclinical development of a rare tumor model: Establishment and characterization of a patient-derived collecting duct carcinoma

Abstract Collecting duct carcinoma (CDC) is a rare type of renal tumor accounting for approximately 2% of all renal cell carcinomas (RCC). Unlike common RCC that arises from the convoluted tubules of the renal cortex, this aggressive tumor is derived from the renal medulla, possibly from the distal collecting duct of Bellini. Clinically this aggressive tumor is shown to have a poor response to therapy and the majority of patients when diagnosed are in the metastatic phase. While several agents have been recently approved for standard RCC, lack of useful preclinical models of CDC have limited drug screening for potential therapies. To this end we have established a patient-derived model of CDC and characterized mutational status and target expression. In addition, we have evaluated in vivo drug sensitivity to agents approved for standard RCC including sorafenib and sunitinib as well as the chemotherapies irinotecan and gemcitabine and targeted therapy temsirolimus. For model establishment a core biopsy was collected and engrafted into an immune-deficient mouse; once take was confirmed tumor tissue was passaged until model stabilization at which point tissue was taken for characterization and in vivo drug sensitivity evaluated. For model characterization, mutation status and expression of target molecules in proliferation pathways were examined by PCR and immunohistochemistry. In the drug sensitivity study, targeted RCC therapies and gemcitabine reported modest tumor growth inhibition while irinotecan was active with significant inhibitory effects. Some activity was reported with the mTOR inhibitor temsirolimus but its effect was not found statistically significant. Overall we have established and characterized a rare patient-derived tumor model of RCC and demonstrated its use as a tool for screening potentially useful agents for patients with collecting duct carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4426. doi:10.1158/1538-7445.AM2011-4426

A study of antifungal drug sensitivity of Candida isolated from human immunodeficiency virus infected patients in Chennai, South India

Background:The purpose of this study was to study the drug sensitivity pattern of Candida seen in HIV seropositive patients in Chennai, South India.Materials and Methods:36 oral rinse samples were collected from HIV seropositive individuals with (21 patients) and without (15 patients) clinical candidiasis. The type of Candidiasis, quantitative estimation, differentiation of candida species and antifungal susceptibility testing was done using different tests.Results:In the 21 patients with candidiasis, pseudomembranous type predominated with low CD4 counts and high colony forming units. Antifungal Drug sensitivity test revealed resistance to fluconazole which is attributed to long term exposure to the drug.Conclusion:The results of the study confirm the hypothesis that candidal species can be isolated in HIV positive patients with clinical candidiasis. In HIV infection there are fluconazole resistant candida species emerging mainly due to long term exposure to the drug.

In-vitro culture of Plasmodium falciparum: Utility of modified (RPNI) medium for drug-sensitivity studies using SYBR Green I assay

Studies were carried out to establish the potential of RPNI medium for drug-sensitivity studies using the MSF assay. The drug sensitivity of standard anti-malarials was compared using both the ( 3H) Hypoxanthine incorporation assay and the MSF assay. The media supplements used during the study have been human serum, FBS and ALBUMAX-II. Drug sensitivity of two parasite lines, adapted to grow separately in conventional as well as in RPNI medium was compared to observe the effect of RPNI medium on functional characteristics of the parasite. The results revealed identical IC 50 values of standard anti-malarials obtained by both the ( 3H) Hypoxanthine incorporation assay and the MSF assay and no untoward effect of FBS and ALBUMAX-II could be noticed on the chemo-sensitivity of standard anti-malarials. Apart from this the chemo-sensitive response of parasite line adapted to grow in RPNI medium was observed to be intact. These findings showed that RPNI medium has potential to be used for chemo-sensitivity studies and the MSF assay being more convenient was observed to be most suitable assay for bio evaluation of new molecules.

Infectious caused by community-acquired Methicillin-Resistant Staphylococcus aureus (CA-MRSA): three-years experience of an universitary hospital in Rome

To date methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common pathogens causing nosocomial infections(1). In Europe the proportion of MRSA is increasing sharply and the distribution varies from country to country. In recent years there has, in various parts of the world, the emergence of infection with strains of S. aureus methicillin-resistant community-acquired (CA-MRSA) than those circulating in hospitals(2). These strains contain a gene that confers resistance to methicillin (mec A SSC mec IV) which is usually associated with the gene for Leukocidin Panton Valentine (PVL) toxin responsible for necrosis of skin and soft tissue (3). In 2006-2008, at the Laboratory of Bacteriology PolyclinicTor Vergata,were isolated a total of 738 strains of S. aureus from biological samples of different nature (oral, vaginal secretions, wound swab, secreted headset, etc ...) of patients related to our surgeries.The identification and study of drug sensitivity of strains were performed with the automatic VITEK2 (bioMérieux). Of the 738 strains of S. aureus identified 212 (28.7%) were resistant to methicillin (MRSA), with an increasing trend over the years: 46 isolates, respectively, in 2006, 76 in 2007 and 90 in 2008. The highest frequency of MRSA (varying between 85% and 95%) was detected in wound swabs from the dispensary and diabetes (diabetic foot).

Pattern of retinoblastoma pathway inactivation dictates response to CDK4/6 inhibition in GBM

Glioblastoma multiforme (GBM) is a fatal primary brain tumor harboring myriad genetic and epigenetic alterations. The recent multidimensional analysis of the GBM genome has provided a more complete view of the landscape of such alterations and their linked pathways. This effort has demonstrated that certain pathways are universally altered, but that the specific genetic events altered within each pathway can vary for each particular patient's tumor. With this atlas of genetic and epigenetic events, it now becomes feasible to assess how the patterns of mutations in a pathway influence response to drugs that are targeting such pathways. This issue is particularly important for GBM because, in contrast to other tumor types, molecularly targeted therapies have failed to alter overall survival substantially. Here, we combined functional genetic screens and comprehensive genomic analyses to identify CDK6 as a GBM oncogene that is required for proliferation and viability in a subset of GBM cell lines and tumors. Using an available small molecule targeting cyclin-dependent kinases (CDKs) 4 and 6, we sought to determine if the specific pattern of retinoblastoma pathway inactivation dictated the response to CDK4/6 inhibitor therapy. We showed that codeletion of CDKN2A and CDKN2C serves as a strong predictor of sensitivity to a selective inhibitor of CDK4/6. Thus, genome-informed drug sensitivity studies identify a subset of GBMs likely to respond to CDK4/6 inhibition. More generally, these observations demonstrate that the integration of genomic, functional and pharmacologic data can be exploited to inform the development of targeted therapy directed against specific cancer pathways.

Abstract 4483: Establishing patient-derived colorectal cancer stem cell models with a PIK3CA mutation for the development of inhibitory drugs as targeted therapies

Abstract Emerging evidences suggest that cancer stem cells (CSC) may be critically responsible for tumor initiation, progression, metastasis, and drug resistance. It becomes important to ask whether anti-cancer agents are able to target the tumor-initiating subpopulation in relevant CSC models. In this study, we first established xenograft tumors in NOD/SCID mice from a colorectal cancer patient specimen and demonstrated that CD133/EpCAM-expressing CSC population was highly tumorigenic. We then sought to propagate the CSC population under a serum-free condition. In culture, the tumor cells formed non-adherent, 3-dimensional spheroids, a fraction of which retained expression of the CSC markers. When exposed to a serum-containing medium, tumor spheroid cells differentiated into epithelial-like adherent cells with an increase in cell proliferation rate. In comparison with the differentiated progeny, tumor spheroid cells exhibited resistance to the standard-of-care agent oxaliplatin and, in limiting dilution assays in mice, displayed substantially higher tumorigenic potential. In contrast to the tumors originated from the differentiated cells, tumor spheroid cell-derived tumors recapitulated not only the CSC frequency marked by CD133/EpCAM expression, but also the histological characters of the original tumor. Similarly, only were the fragments of spheroid cell-derived xenograft tumors capable of regenerating highly proliferative tumors in secondary transplantation. Thus, the tumor spheroid culture is indeed enriched of drug resistant, self-renewing, and tumor-initiating CSC populations. Mutation profiling of frequently mutated oncogenes using Sequenom OncoCarta™ panel identified a mutation in the kinase domain of PIK3CA (H1047R) in the cultured CSCs. This mutation has been reported present in a large number of colon cancer patients and likely functions as an oncogene (Samuels et al., Science 304:554; 2004). We further demonstrated that a dual mammalian target of rapamycin (mTOR)/phosphoinositide 3-kinase (PI3K) inhibitor (PF-04691502) exhibited a more potent effect on inhibition of in vitro proliferation of the mutated CSCs compared to the chemotoxic agent oxaliplatin. Collectively, our findings suggest that CSC models provide a novel avenue to drug sensitivity and efficacy studies. The well-characterized CSC model systems may assist in the development of more effective therapy against the subpopulation of tumors driven by the CSCs bearing specific mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4483.

The butyrylcholinesterase knockout mouse a research tool in the study of drug sensitivity, bio-distribution, obesity and Alzheimer's disease

Butyrylcholinesterase (BChE) mutations common in the human population may result in complete or partial BChE deficiency, making the BChE knockout (KO) mouse a model for human deficiencies. The BChE KO mouse cannot tolerate standard doses of the muscle relaxant succinylcholine or the Alzheimer's disease drugs huperzine A and donepezil. It is resistant to the asthma drug bambuterol. The importance of BChE in detoxication of cocaine has been demonstrated by hepatotoxicity and cardiotoxicity in cocaine-challenged BChE KO mice. The BChE KO mouse becomes obese on a high-fat diet, suggesting a role for BChE in fat metabolism. BChE serves as a backup for acetylcholinesterase by hydrolyzing the neurotransmitter acetylcholine in acetylcholinesterase knockout mice. Imaging studies show that BChE injected intrathecally crosses the blood–brain barrier. Mice, but not humans, have carboxylesterase in their blood. Carboxylesterase obscures the role of BChE in detoxication of organophosphorus pesticides. Future studies will make a double knockout that has neither BChE nor carboxylesterase. The double knockout is expected to be unusually sensitive to the toxicity of organophosphorus pesticides. Knowledge of drug sensitivities in the mouse model of human BChE deficiency will aid in understanding adverse drug effects in humans.

Expression of c-Kit isoforms in multiple myeloma: differences in signaling and drug sensitivity

c-Kit is expressed in the plasma cells from 30% of patients with multiple myeloma. Two different isoforms of c-Kit, characterized by the presence or absence of the tetrapeptide sequence GNNK in the extracellular domain, have been described. However, their expression and function in myeloma cells are unknown. We explored the function and expression of these c-Kit isoforms in myeloma cells. Expression of c-Kit isoforms was investigated by reverse transcriptase polymerase chain reaction in fresh plasma cells from patients and cell lines. The function of these c-Kit isoforms was analyzed upon expression in myeloma cells. Signaling was investigated by western blotting using antibodies specific for activated forms of several signaling proteins. The impact of c-Kit on the action of drugs commonly used in the treatment of multiple myeloma was investigated by MTT proliferation assays. Fresh plasma cells from patients as well as myeloma cell lines expressed the two isoforms of c-Kit. Retroviral infection of myeloma cells with vectors that code for c-Kit-GNNK+ or c-Kit-GNNK- forms demonstrated differences in the kinetics of phosphorylation between these isoforms. Stem cell factor-induced activation of the GNNK- form was faster and more pronounced than that of the GNNK+ form, whose activation, however, lasted for longer. The c-Kit receptors weakly activated the Erk1/2 and Erk5 pathways. Both receptors, however, efficiently coupled to the PI3K/Akt pathway, and stimulated p70S6K activation. The latter was sensitive to the mTOR inhibitor, rapamycin. Studies of drug sensitivity indicated that cells expressing the GNNK- form were more resistant to the anti-myeloma action of bortezomib and melphalan. Our data indicate that c-Kit expression in multiple myeloma cells is functional, and coupled to survival pathways that may modulate cell death in response to therapeutic compounds used in the treatment of this disease.

Alterations in γ-Actin and Tubulin-Targeted Drug Resistance in Childhood Leukemia

Proteomic investigations have revealed alterations in cytoskeletal proteins expressed in human acute lymphoblastic leukemia cells that are resistant to microtubule-disrupting agents. We characterized gamma-actin expression in antimicrotubule drug-resistant leukemia and examined the effect of altered gamma-actin in resistance of acute lymphoblastic leukemia to antimicrotubule agents. Two-dimensional polyacrylamide gel electrophoresis and mass spectrometry were used to identify actin proteins in human acute lymphoblastic leukemia cell lines resistant to vinblastine (CCRF-CEM/VLB100 cells) and desoxyepothilone B (CCRF-CEM/dEpoB140 cells). Fluorescence-based cycle sequencing was used to detect gene mutations. Site-directed mutagenesis was used to generate mutant gamma-actin expression plasmids, which were used to transfect mouse NIH/3T3 cells. Clonogenic analysis was used for drug sensitivity studies. A small interfering RNA (siRNA) was used to block gamma-actin gene expression in human neuroblastoma SH-EP cells. Expression of gamma-actin (normalized to that of beta2-microglobulin [beta2M]) in primary leukemia cells obtained from patients at diagnosis (n = 44) and relapse (n = 25) was examined using semiquantitative reverse transcription-polymerase chain reaction. Statistical significance of changes in the ratio of gamma-actin to beta2M expression between diagnosis and relapse samples was determined by two-sided unpaired Student's t tests. We identified novel mutant forms of gamma-actin and the concomitant loss of wild-type gamma-actin in CCRF-CEM/VLB100 cells and CCRF-CEM/dEpoB140 cells. Mouse NIH/3T3 cells that expressed the mutant gamma-actin proteins were more resistant to antimicrotubule agents than cells transfected with empty plasmid. Human neuroblastoma SH-EP cells transfected with gamma-actin siRNA displayed higher relative resistance to paclitaxel (P<.001), vinblastine (P = .04), and epothilone B (P = .045) than mock-transfected cells. No gamma-actin gene mutations were identified in 37 samples of primary leukemia cells (eight from patients at diagnosis, 29 from patients at relapse). Gamma-actin gene expression was lower in acute lymphoblastic leukemia samples collected at clinical relapse (n = 25; mean gamma-actin/beta2M = 0.53) than in samples collected at diagnosis (n = 44; mean gamma-actin/beta2M = 0.68; difference = 0.15, 95% confidence interval [CI] = 0.04 to 0.27, P = .01). These data provide functional and associative clinical evidence of a novel form of drug resistance that involves interactions between gamma-actin and microtubules.

A randomized trial comparing the efficacy of four treatment regimens for uncomplicated falciparum malaria in Assam state, India

A four-arm drug sensitivity study compared chloroquine, sulfadoxine-pyrimethamine (SP), mefloquine and mefloquine-artesunate in Sonitpur and Karbi Anglong districts in Assam state, India. Two criteria were used to ascertain outcome: success of clinical treatment and parasitologic cure. In Sonitpur, at 14 days, there were 36/56 early and late treatment failures plus late parasitologic failures to chloroquine and 16/56 for SP. In Karbi Anglong, combined treatment failure at 14 days was 16/56 to chloroquine and 8/60 to SP. Mefloquine and mefloquine-artesunate demonstrated 93.9% and 93.6% sustained responses respectively at 42 days. High failure rates to both chloroquine and SP preclude the use of these drugs as first-line treatment for uncomplicated falciparum malaria in this region. A mefloquine-artesunate combination presents an effective alternative utilizing the currently recommended higher dose of mefloquine.

Avicins: A Novel Class of Anti-Myeloma Agents.

Multiple myeloma (MM) remains an incurable neoplasia, despite recent development of several novel therapies. As part of our efforts to identify new compounds with anti-MM activity, we evaluated the class of avicins, which are triterpenoid saponins that have been shown to induce apoptosis of neoplastic cells by affecting mitochondrial function independently of membrane-bound death receptors. Because we have previously shown that mitochondria constitute key regulators of MM cell responsiveness to diverse anti-tumor agents, (e.g. the proteasome inhibitor bortezomib), we evaluated the in vitro anti-MM effects of this class of compounds. Our vitro drug-sensitivity studies showed that Avicin D and Avicin G, the main members of this class of compounds, are active against a broad panel of MM cell lines and primary tumor cells, including tumor cells resistant to conventional (e.g. dexamethasone, alkylating agents, anthracyclines) or novel (e.g. thalidomide, immunomodulatory thalidomide derivatives, proteasome inhibitor PS-341[bortezomib], Apo2L/TRAIL) anti-MM agents. Using MTT survival assays, we confirmed that the IC50 values for both Avicins were highly concordant and were less than 250 nM for the overwhelming majority of MM cell lines tested. Importantly, this potent in vitro anti-MM activity was triggered by concentrations of Avicins which had minimal, if any, effect on the viability of normal hematopoietic cells or bone marrow stromal cells. Furthermore these IC50 values were comparable with the in vitro activity of this agent among the most Avicin-sensitive tumor models that have been previously tested. This potent anti-MM effect was not inhibited by transfection of MM cells with construct for constitutively active Akt. Although cytokine- or cell adhesion-mediated interactions of the local bone marrow (BM) microenvironment (e.g. BM stromal cells) protects MM cells from conventional therapies (e.g. dexamethasone or cytotoxic chemotherapy), avicins were able to overcome this protective effect in co-culture models of MM cells with BM stromal cells and sensitized MM cells to cytotoxic chemotherapy-induced cell death. Using hierarchical clustering analyses and relevance network algorithms, we compared the patterns of MM cell sensitivity to Avicin D vs Avicin G vs. other anti-cancer drugs and found that the pattern of dose-response relationships of the 2 main members of this class of compounds are very similar to each other, but clearly distinct from the patterns of sensitivity or resistance to other drugs, either conventional or investigational. This further supports the notion that the anti-MM properties of Avicins are mediated by molecular mechanisms distinct from those of currently available anti-MM drugs, and also suggests that Avicins may have anti-tumor activity even against subgroups of MM which may be resistant to other novel therapies that are currently in clinical development. These results have provided the framework for ongoing in vivo studies of anti-tumor activity of these agents, to evaluate the feasibility of future clinical trials of Avicins to improve patient outcome in MM.

Differential gene expression analysis by DNA microarrays technology and its application in molecular oncology

Accumulation of genetic and epigenetic aberrations leads to malignant transformation of normal cells. Functional studies of cancer using genomic and proteomic tools will help to reveal the true complexity of the processes leading to cancer development in humans. Until recently, diagnosis and prognosis of cancer was based on conventional pathologic criteria and epidemiological evidence. Certain tumors were divided only into relatively broad histological and morphological subcategories. Rapidly developing methods of differential gene expression analysis promote the search for clinically relevant genes changing their expression levels during malignant transformation. DNA microarrays offer a unique possibility to rapidly assess the global expression picture of thousands genes in any given time point and compare the detailed combinatory analysis results of global expression profiles for normal and malignant cells at various functional stages or separate experimental conditions. Acquisition of such "genetic portraits" allows searching for regularity and difference in expression patterns of certain genes, understanding their function and pathological importance, and ultimately developing the "molecular nosology" of cancer. This review describes the basis of DNA microarray technology and methodology, and focuses on their applications in molecular classification of tumors, drug sensitivity and resistance studies, and identification of biological markers of cancer.