Resistance gene expression determines the in vitro chemosensitivity of non-small cell lung cancer (NSCLC).

BACKGROUND: NSCLC exhibits considerable heterogeneity in its sensitivity to chemotherapy and similar heterogeneity is noted in vitro in a variety of model systems. This study has tested the hypothesis that the molecular basis of the observed in vitro chemosensitivity of NSCLC lies within the known resistance mechanisms inherent to these patients' tumors. METHODS: The chemosensitivity of a series of 49 NSCLC tumors was assessed using the ATP-based tumor chemosensitivity assay (ATP-TCA) and compared with quantitative expression of resistance genes measured by RT-PCR in a Taqman Array following extraction of RNA from formalin-fixed paraffin-embedded (FFPE) tissue. RESULTS: There was considerable heterogeneity between tumors within the ATP-TCA, and while this showed no direct correlation with individual gene expression, there was strong correlation of multi-gene signatures for many of the single agents and combinations tested. For instance, docetaxel activity showed some dependence on the expression of drug pumps, while cisplatin activity showed some dependence on DNA repair enzyme expression. Activity of both drugs was influenced more strongly still by the expression of anti- and pro-apoptotic genes by the tumor for both docetaxel and cisplatin. The doublet combinations of cisplatin with gemcitabine and cisplatin with docetaxel showed gene expression signatures incorporating resistance mechanisms for both agents. CONCLUSION: Genes predicted to be involved in known mechanisms drug sensitivity and resistance correlate well with in vitro chemosensitivity and may allow the definition of predictive signatures to guide individualized chemotherapy in lung cancer.

Enzyme-linked immunomagnetic chemiluminescent detection of Escherichia coli O157:H7.

E. coli O157:H7 is a pathogenic microorganism that has been implicated in numerous cases of foodborne illnesses. A variety of rapid methods exist that show promise for the presumptive detection of this pathogen without the immediate need for incubating test samples for hours to days in microbial enrichment and culture media. In recent years, highly sensitive chemiluminescence has become a more affordable and portable detection method. Chemiluminescent detection has been coupled with the selectivity of antibodies, magnetic microparticle separation/isolation, and enzymatic signal amplification in order to develop a rapid method, termed enzyme-linked immunomagnetic chemiluminescence (ELIMCL). This work presents the application of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline with a detection limit of 7.6 x 10(3) for live cells in approx. 75 min assay time. The blocking agent casein and the surfactant Tween 20 were used to lower background luminescence and thus maximize signal-to-noise ratios. After 5.5 h of enrichment culture, ELIMCL was demonstrated to detect E. coli O157:H7 inoculated in ground beef at 10 CFU/g in a total assay time of about 7 h.

Immunoassay of infectious agents.

Immunoassays have evolved for a broad range of applications since the pioneering work of Yalow and Berson who developed the first competitive radioimmunoassay (RIA) for human insulin in 1959. Immunoassay detection of specific antigens and host-produced antibodies directed against such antigens consitutes one of the most widely used and successful methods for diagnosing infectious diseases (IDs). The number and variety of new assay systems that are continually being developed reflect the increasing demand for immunoassays possessing greater sensitivity, speed, and ease of use. This trend has been driven, in part, by the need for improved immunodiagnostic systems to perform rapid testing and counter emerging IDs and biothreat (BT) agents. Another factor driving this trend is the need to integrate immunoassays with more sensitive nucleic acid-based methods for a comprehensive approach. Here we examine the development of immunoassays, some of the key formats used for the detection and identification of BT/ID agents, and the application of these technologies under different scenarios.

Luminescence applications for chemotherapeutic drug development.

The application of in vitro and in vivo ATP bioluminescence systems as an integrated approach for preclinical research and development of new chemotherapeutic drugs is described. This approach includes both (a) the in vitro tumor response assay (TRA) system that utilizes new technologies for cell culture and ATP measurement of clinical specimens and (b) the use of human tumor cell lines transfected with Photinus pyralis luciferase (luc) gene for both in vitro and in vivo studies. Dried reagent microplates for TRA culture and counting procedures are described for a two-stage TRA method, which can be used to evaluate drug sensitivity and resistance of cells from clinical specimens after initial drug exposure in vitro. The use of dried reagent counting plates for screening and testing of agents against tumor cell lines is described, as well as an alternative method for screening and testing chemotherapeutic drugs in vitro with luc-transfected human tumor cell lines. The potential application of luc-transfected reporter cell lines for in vivo studies of drug activity with photon imaging for analysis is discussed.

Chemosensitivity of normal human trophoblasts evaluated by a newly developed ATP-based luminescence assay.

Trophoblast injury may be one of the possible causes of fetal distress associated with chemotherapy administered during pregnancy. The purpose of this study was to investigate the ex vivo chemosensitivity of normal trophoblasts (NTB) against commonly used antineoplastic agents. Using the newly developed ex vivo ATP-based trophoblast assay (ATP-TBA), 31 NTB freshly sampled from human placentas (gestational week 7-42) were tested against dactinomycin (Act-D), 5-fluorouracil (5-FU), 4-OOH-cyclophosphamide (4-HC), vincristine (VCR) and methotrexate (MTX) alone or in combination with calcium folate (LV). All agents were studied at concentrations relevant to clinical dosages normally used for chemotherapy of solid neoplasms. Of 31 samples studied with the ATP-TBA, 20 (65%) were evaluable. VCR, Act-D and 4-HC were the most active drugs with 55, 45 and 45% of samples responding ex vivo. Antimetabolites were less active, producing ex vivo response rates of 25 (MTX) and 20% (5-FU), respectively. MTX activity was largely neutralized by adding LV. The chemosensitivity of NTB showed considerable inter-individual variations and did not decrease with increasing gestational age. We therefore conclude that NTB of any gestational age exhibit considerable ex vivo sensitivity against common anticancer agents which is comparable to that observed for various solid tumors. The ATP-TBA may be helpful in planning future trials with both single agents and drug combinations in order to standardize and optimize chemotherapy during pregnancy.