In vitro sensitivity assays in cancer: a review, analysis, and prognosis

Tumors are complex systems consisting of heterogeneous cancer cells as well as normal cells with each exhibiting unique drug sensitivity spectra. There have been many attempts to design in vitro systems to determine drug response to tumors. The most widely used system is the clonogenic assay. which has demonstrated some clinical predictivity. However, the clonogenic assay has been shown to have negative aspects, including low frequency of evaluation, clump artifacts, lack of cytotoxic end-points and disruption of normal cell-cell interactions existing in a true tissue environment. Newer models are described utilizing cytotoxic as well as cell-proliferation end-points, and maintenance of three-dimensional tissue architecture in vitro. It is concluded that less artifactual, more realistic models can be used to select more tumor-specific drugs which themselves in turn will make in vitro chemosensitivity assays more useful for cancer patients.     

High-throughput assays using a luciferase-expressing replicon, virus-like particles, and full-length virus for West Nile virus drug discovery

Many flaviviruses cause significant human disease worldwide. The development of flavivirus chemotherapy requires reliable high-throughput screening (HTS) assays. Although genetic systems have been developed for many flaviviruses, their usage in antiviral HTS assays has not been well explored. Here we compare three cell-based HTS assays for West Nile virus (WNV) drug discovery: (i) an assay that uses a cell line harboring a persistently replicating subgenomic replicon (containing a deletion of viral structural genes), (ii) an assay that uses packaged virus-like particles containing replicon RNA, and (iii) an assay that uses a full-length reporting virus. A Renilla luciferase gene was engineered into the replicon or into the full-length viral genome to monitor viral replication. Potential inhibitors could be identified through suppression of luciferase signals upon compound incubation. The antiviral assays were optimized in a 96-well format, validated with known WNV inhibitors, and proved useful in identifying a new inhibitor(s) through HTS of a compound library. In addition, because each assay encompasses multiple but discrete steps of the viral life cycle, the three systems could potentially be used to discriminate the mode of action of any inhibitor among viral entry (detected by assays ii and iii but not by assay i), replication (including viral translation and RNA synthesis; detected by assays i to iii), and virion assembly (detected by assay iii but not by assays i and ii). The approaches described in this study should be applicable to the development of cell-based assays for other flaviviruses.     

A duplexed phenotypic screen for the simultaneous detection of inhibitors of the molecular chaperone heat shock protein 90 and modulators of cellular acetylation

Histone deacetylases (HDACs), histone acetyltransferases (HATs), and the molecular chaperone heat shock protein 90 (HSP90) are attractive anticancer drug targets. High-throughput screening plays a pivotal role in modern molecular mechanism-based drug discovery. Cell-based screens are particularly useful in that they identify compounds that are permeable and active against the selected target or pathway in a cellular context. We have previously developed time-resolved fluorescence cell immunosorbent assays (TRF-Cellisas) for compound screening and pharmacodynamic studies. These assays use a primary antibody to the single protein of interest and a matched secondary immunoglobulin labeled with an europium chelate (Eu). The availability of species-specific secondary antibodies labeled with different lanthanide chelates provides the potential for multiplexing this type of assay. The approach has been applied to the development of a 384-well duplexed cell-based screen to simultaneously detect compounds that induce the co-chaperone HSP70 as a molecular marker of potential inhibitors of HSP90 together with those that modulate cellular acetylation (i.e., potential inhibitors of histone deacetylase or histone acetyltransferase activity). The duplexed assay proved reliable in high-throughput format and approximately 64,000 compounds were screened. Following evaluation in secondary assays, 3 of 13 hits from the HSP70 arm were confirmed. Two of these directly inhibited the intrinsic ATPase activity of HSP90 whereas the third seems to have a different mechanism of action. In the acetylation arm, two compounds increased cellular acetylation, one of which inhibited histone deacetylase activity. A third compound decreased cellular histone acetylation, potentially through a novel mechanism of action.     

Malaria drug-susceptibility testing. HRP2-based assays: current data, future perspectives

In past decades, malaria in-vitro drug-susceptibility assays have become an indispensable tool for the development of novel drugs, as well as for the surveillance of antimalarial drug resistance. The traditional in-vitro assays, however, remain tedious procedures, which, depending on the method employed, require a high degree of expertise, sophisticated laboratory infra-structure, skills and patience. We therefore developed a new drug sensitivity assay for Plasmodium falciparum based on the measurement of histidine-rich protein II (HRP2), a histidine and alanine-rich protein produced by P. falciparum. The assay uses a simple HRP2 double-site sandwich ELISA to quantify parasite growth and its inhibition. The complete ELISA takes about 2-3 hours to perform and requires little technical equipment. In our experiments with laboratory strains of P. falciparum against common antimalarials, the results closely parallel those obtained from the isotope assay and from WHO schizont maturation tests (P < 0.001). Preliminary results using the HRP2 assay in our field studies in 2002 suggest similar outcomes. In this setting the high sensitivity and simplicity of the assay is of particular advantage. The data closely parallel those obtained with the traditional WHO assay (Mean difference on the log scale: 0.033; R = 0.942; P < 0.001). The assay is currently being further validated under field conditions. It has proved to be a valuable tool for a wide range of applications, from epidemiological field studies to the screening of new drugs, and may therefore have the potential to replace traditional in-vitro drug-sensitivity techniques.     

MTT法测定复发转移性乳腺癌体外化疗药物敏感性实验研究

目的探讨MTT法体外药敏试验在复发转移性乳腺癌化疗中的价值。方法取35例复发转移性乳腺癌患者肿瘤组织，简易机械法制备细胞悬液，应用MTT法体外药敏试验测定其对10种常用化疗药物的敏感性，并指导临床用药，同时与临床疗效进行相关分析。结果MTT法检测复发转移乳腺癌患者药敏试验的成功率为91．4％，体外药敏试验的敏感性为86．4％，特异性为80．0％，阳性预测值为90．5％，阴性预测值为72．7％，总的预测准确率为84．4％。结论MTT法可作为临床上指导复发转移乳腺癌个体化化疗的有效方法。     

Use of marine toxins in combination with cytotoxic drugs for induction of apoptosis in acute myelogenous leukaemia cells

Intensive chemotherapy for acute myelogenous leukaemia (AML) results in an overall long-term disease-free survival of < 50%. This percentage reflects an improved survival for certain subsets of patients with low-risk cytogenetic abnormalities after treatment with high-dose cytarabine, whereas lower long-term survival is seen for other patients and especially for the large group of elderly patients. New treatment strategies are therefore considered in AML and one approach is to target the regulation of apoptosis in AML cells with new pharmacological agents. Regulation of apoptosis seems to be clinically important in AML as intracellular levels of apoptosis-regulating mediators can be used as predictors of prognosis in AML. It is also well documented that cytotoxic drugs exert important antileukaemic effects through induction of apoptosis. Marine toxins represent new pharmacological agents with proapoptotic effects and should be considered for combination therapy with cytotoxic drugs. These agents are already useful laboratory tools for in vitro studies of AML cells but it is still too early to conclude whether they will become useful in clinical therapy. One of the major problems to be investigated is the toxicity of combination therapy, although this may be solved by the coupling of toxins to antibodies or growth factors with a preferential binding to AML cells. Other problems that have to be addressed are the possible effect of the toxins’ tumour promoting effects on chemosensitivity in relapsed AML and the possibility of cross-resistance between cytotoxic drugs and toxins.     

Use of marine toxins in combination with cytotoxic drugs for induction of apoptosis in acute myelogenous leukaemia cells

Intensive chemotherapy for acute myelogenous leukaemia (AML) results in an overall long-term disease-free survival of < 50%. This percentage reflects an improved survival for certain subsets of patients with low-risk cytogenetic abnormalities after treatment with high-dose cytarabine, whereas lower long-term survival is seen for other patients and especially for the large group of elderly patients. New treatment strategies are therefore considered in AML and one approach is to target the regulation of apoptosis in AML cells with new pharmacological agents. Regulation of apoptosis seems to be clinically important in AML as intracellular levels of apoptosis-regulating mediators can be used as predictors of prognosis in AML. It is also well documented that cytotoxic drugs exert important antileukaemic effects through induction of apoptosis. Marine toxins represent new pharmacological agents with proapoptotic effects and should be considered for combination therapy with cytotoxic drugs. These agents are already useful laboratory tools for in vitro studies of AML cells but it is still too early to conclude whether they will become useful in clinical therapy. One of the major problems to be investigated is the toxicity of combination therapy, although this may be solved by the coupling of toxins to antibodies or growth factors with a preferential binding to AML cells. Other problems that have to be addressed are the possible effect of the toxins' tumour promoting effects on chemosensitivity in relapsed AML and the possibility of cross-resistance between cytotoxic drugs and toxins.     

Chemosensitivity profile of cancer cell lines and identification of genes determining chemosensitivity by an integrated bioinformatical approach using cDNA arrays

We have established a panel of 45 human cancer cell lines (JFCR-45) to explore genes that determine the chemosensitivity of these cell lines to anticancer drugs. JFCR-45 comprises cancer cell lines derived from tumors of three different organs: breast, liver, and stomach. The inclusion of cell lines derived from gastric and hepatic cancers is a major point of novelty of this study. We determined the concentration of 53 anticancer drugs that could induce 50% growth inhibition (GI50) in each cell line. Cluster analysis using the GI50s indicated that JFCR-45 could allow classification of the drugs based on their modes of action, which coincides with previous findings in NCI-60 and JFCR-39. We next investigated gene expression in JFCR-45 and developed an integrated database of chemosensitivity and gene expression in this panel of cell lines. We applied a correlation analysis between gene expression profiles and chemosensitivity profiles, which revealed many candidate genes related to the sensitivity of cancer cells to anticancer drugs. To identify genes that directly determine chemosensitivity, we further tested the ability of these candidate genes to alter sensitivity to anticancer drugs after individually overexpressing each gene in human fibrosarcoma HT1080. We observed that transfection of HT1080 cells with the HSPA1A and JUN genes actually enhanced the sensitivity to mitomycin C, suggesting the direct participation of these genes in mitomycin C sensitivity. These results suggest that an integrated bioinformatical approach using chemosensitivity and gene expression profiling is useful for the identification of genes determining chemosensitivity of cancer cells.     

Current opinions on therapeutic drug monitoring of immunosuppressive drugs

The pharmacokinetics of the immunosuppressive drugs cyclosporine, tacrolimus, mycophenolate mofetil (MMF), and sirolimus are complex and unpredictable. A narrow therapeutic index unique to each patient, as well as variable absorption, distribution, and elimination, are characteristics of these drugs. Therapeutic drug monitoring plays a key role in helping clinicians maintain blood and plasma levels of immunosuppressive drugs within their respective therapeutic ranges. Variation in concentrations outside the narrow therapeutic ranges can result in adverse clinical outcomes. Therapeutic drug monitoring ensures that concentrations are not too high or too low, thereby reducing the risks of toxicity or rejection, respectively. Therapeutic monitoring of immunosuppressive drugs has been based on several choices of assay and biologic fluid (i.e., whole blood, plasma) appropriate for a particular drug. High-performance liquid chromatography (HPLC) remains the gold standard among assay methods used to monitor immunosuppressive drugs. Although HPLC is the assay of choice for cyclosporine, newer monoclonal assays are suitable as well for routine monitoring. HPLC is also widely used for therapeutic drug monitoring of mycophenolic acid, the active metabolite of MMF, and an immunoassay (used in European centers) has been developed. Therapeutic drug monitoring of tacrolimus has been improved with the recent development of assays with greater sensitivity and specificity for tacrolimus than those previously available. No commercial assays are currently available for the therapeutic monitoring of sirolimus. It is also important to identify a specific pharmacokinetic parameter for each individual drug, whether it is trough or area under the concentration-time curve, that may be most useful as a tool for optimal therapeutic drug monitoring in clinical practice. With an increased understanding of the pharmacokinetics of immunosuppressive drugs, therapeutic drug monitoring guidelines will be more clearly defined to ensure the safe and effective management of transplant recipients.     

Histidine-rich protein II: a novel approach to malaria drug sensitivity testing

The production of histidine-rich protein II (HRP2), a histidine- and alanine-rich protein produced by Plasmodium falciparum, is closely associated with the development and proliferation of the parasite and therefore is perfectly suited to reflect growth inhibition as a measure of drug susceptibility. It was the aim of the present study to develop a malaria drug sensitivity assay based on the measurement of HRP2 in a simple enzyme-linked immunosorbent assay (ELISA). The new test proved to be as reliable as traditional in vitro assays, while it was considerably easier to establish and perform. Parasites are incubated at an initial level of parasitemia of 0.01 to 0.1% on microculture plates predosed with ascending concentrations of antimalarial drugs. After incubation for 48 to 72 h, the samples are freeze-thawed and transferred to ELISA plates. The complete ELISA takes about 2.5 h to perform, may be carried out with commercially available test kits, and requires relatively little technical equipment. In correlation analysis, the results closely paralleled those obtained by the isotopic assay (R = 0.892; P < 0.0001) and World Health Organization schizont maturation tests (R = 0.959; P < 0.0001). The novel HRP2 drug susceptibility assay proved to be very sensitive, simple to establish, and highly reproducible. It can be used for a wide range of applications, from epidemiological studies to the screening of new drugs, and may have the potential to replace traditional in vitro techniques. Standard operating procedures, updated information, and analytical software are available from http://malaria.farch.net.     

Cloning and sequencing an unknown gene of Tropheryma whipplei and development of two LightCycler PCR assays

Whipple's disease is a rare infectious illness that can affect any organ system in the body. It is caused by Tropheryma whipplei, a Gram-positive rod-shaped bacterium with a high G + C content, classified within the actinobacteria. For decades, laboratory detection has been based on microscopy and the periodic acid-Schiff (PAS) staining of biopsies. Recently, PCR has become a useful tool to detect T. whipplei DNA in various clinical specimens. However, a positive PCR result does not confirm Whipple's disease as it has been shown that asymptomatic persons can harbor T. whipplei DNA. Since there is not yet much known about the genome of T. whipplei, genome-walking represents a convenient method to determine unknown gene sequences. Starting from a RAPD fragment we have sequenced and cloned an open reading frame (ORF) of 843 bp. Two real-time PCR assays targeting the ORF fragment and the 16S rRNA gene, respectively, were developed. Compared to a conventional 16S rRNA PCR system the ORF LightCycler assay proved to be very specific (100%) but not sufficiently sensitive (62.4%). In contrast, the 16S rRNA LightCycler assay showed a sensitivity of 95.7% and a specificity of 97.8%. Thus, the 16S rRNA gene assay but not that targeting the new ORF is a suitable alternative to conventional PCR methods.     

Interference by anti-cancer chemotherapeutic agents in the MTT-tumor chemosensitivity assay

BACKGROUND: One of the major goals of oncology is to predict the response of patients with cancer to chemotherapeutic agents by employing laboratory methods variously called 'tumor chemosensitivity assays', 'drug response assays', or 'drug sensitivity assays', in vitro. The MTT assay is one of the methods used to predict the drug response in malignancies. However, it may suffer from interference by the anticancer drugs with the MTT assay. METHODS: The MTT assay, a colorimetric viability assay, was checked in a cell-free system in terms of its possible chemical interactions with 22 different anticancer drugs. RESULTS: It was found that epirubicine, paclitaxel, doxetaxel, and cisplatin caused a relatively significant increase in absorbance values, resulting in the MTT assay giving rise to false results (untrue increase in viability) although most of the drugs tested did not seem to cause any significant change. CONCLUSIONS: It was concluded that before employing the MTT assay, drugs (or any kind of substances) to be included in the assay should be checked first in terms of possible chemical interactions with MTT, otherwise it may be impossible to evaluate the MTT viability assay results correctly.     

Sensitive thyroid-stimulating hormone assays: clinical applications and limitations

Sensitive TSH assays have important applications in various conditions, including the diagnosis of hypothyroidism and hyperthyroidism, monitoring thyroid hormone therapy and treated thyrotoxic patients, and evaluating thyroid dysfunction in nonthyroidal illnesses and pregnancy. Interpretation of the TSH value should be made with a clear understanding of its limitations. TSH may be inappropriately secreted by pituitary tumors and by pituitary dysfunction due to thyroxine resistance. At present, it is uncertain whether clinically euthyroid patients with autonomously functioning thyroid nodules, or with multinodular goiters, or patients taking thyroid hormone who have suppressed TSH values, are actually euthyroid at a cellular level. Other factors that affect TSH levels are the biologic variation in its secretion, the presence of heterophilic antibodies in a patient's serum, and various drugs. But perhaps the most important factor affecting the TSH assay is severe nonthyroidal illness in hospitalized patients. The new ultrasensitive TSH assay does not yet replace other thyroid function tests, but it is clearly emerging as an important means of screening patients for thyroid dysfunction, especially ambulatory patients without other serious illnesses. It can usually separate patients with thyroid dysfunction from euthyroid individuals. Good clinical assessment is always necessary, and other thyroid function tests are often needed. The sensitivity of these new TSH assays in the diagnosis of thyrotoxicosis and hypothyroidism is excellent; the specificity is not as good. Nonetheless, at present this test can be used in the initial diagnosis of thyroid dysfunction as outlined in Figure 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

人大肠癌原代培养体外药敏试验的研究

目的探讨用生物荧光肿瘤体外药敏检测技术（ATP－TCA）研究大肠癌药敏的异质性和个体化疗的可行性。方法用ATP—TCA技术检测50份大肠癌标本对16种抗癌药单药或联合用药的敏感性。结果不同标本的药物敏感性存在着明显差异，单药中最有效的药物依次为长春瑞滨、羟基喜树碱、氟尿嘧啶和紫杉醇，敏感率分别为34．1％、31．6％、27．6％和24．3％。最有效联合用药方案是氟尿嘧啶＋丝裂霉素＋阿糖胞苷，敏感率为100％（11／11份），其次是氟尿嘧啶＋顺铂＋阿霉素和健择＋顺铂。结论大肠癌对抗癌药物的敏感程度普遍较低，且存在明显异质性。试验结果与临床治疗经验比较一致，ATP－TCA可用于为大肠癌患者选择合适的化疗药物。     

分化抑制因子1表达对膀胱癌细胞系化疗敏感性及细胞凋亡的影响

目的探讨分化抑制因子1（inhibitor of differentiation,Id-1）下调对膀胱癌细胞系化疗敏感性及药物诱导细胞凋亡的影响。方法选取膀胱上皮癌细胞系MGH-U1,在构建Id-1si-RNA载体后,通过逆转录病毒将其转染至MGH-U1细胞,筛选出稳定低表达Id-1的克隆（si-Id-1）和对照载体克隆（sscon）。MTT法和集落形成实验比较sscon细胞和si-Id-1克隆细胞对表柔比星的敏感性;Ⅰ型胶原侵袭实验证明Id-1下调对膀胱癌细胞侵袭能力的影响;Western blot实验检测凋亡相关蛋白,比较凋亡蛋白表达水平,研究Id-1下调对表柔比星诱导的细胞凋亡的影响。结果 si-Id-1克隆细胞对表柔比星的敏感性显著高于sscon细胞;si-Id-1克隆细胞的侵袭能力明显低于sscon细胞;Id-1下调会诱导凋亡通路激活因子Cleaved PARP和Cleaved Caspase3的表达水平增高,从而证明Id-1下调会增加表柔比星诱导的细胞凋亡。结论膀胱癌细胞Id-1下调可以增加抗肿瘤药物诱导的细胞凋亡,从而增加肿瘤细胞的化疗敏感性。     

Protein phosphorylation and signal transduction.

It is now generally accepted that protein phosphorylation-dephosphorylation has a role in the regulation of essentially all cellular functions. Thus, it is of interest that this process is involved in signal transduction. Nonetheless, the extent to which protein phosphorylation participates in signaling is truly remarkable. Almost every known signaling pathway eventually impinges on a protein kinase, or in some instances, a protein phosphatase. The diversity of these enzymes is noteworthy, and it is of interest that many biotechnology companies are eyeing them as potentially important targets for drugs. Such drugs may have important therapeutic applications, and in any event, they certainly will be useful to investigators who study signal transduction. Indeed, this already has been proven to be true.     

Inhibition of phosphorylation of the colony-stimulating factor-1 receptor (c-Fms) tyrosine kinase in transfected cells by ABT-869 and other tyrosine kinase inhibitors

The properties of several multitargeted receptor tyrosine kinase inhibitors have been studied for their inhibition of colony-stimulating factor-1 receptor (CSF-1R) signaling. A structurally novel, multitargeted tyrosine kinase inhibitor (ABT-869), imatinib (STI571), and four compounds currently in clinical development (AG013736, BAY 43-9006, CHIR258, and SU11248) were tested for inhibition of CSF-1R signaling in both the enzymatic and cellular assays. ABT-869 showed potent CSF-1R inhibition in both the enzyme and cell-based assays (IC50s < 20 nmol/L). In contrast to a previous report, we have found that imatinib has activity against human CSF-1R in both assays at submicromolar concentrations. In enzyme assays, we have found that the inhibition of CSF-1R by both ABT-869 and imatinib are competitive with ATP, with Ki values of 3 and 120 nmol/L, respectively. SU11248 is a potent inhibitor of CSF-1R in the enzyme assay (IC50 = 7 nmol/L) and inhibits receptor phosphorylation in the cellular assay (IC50 = 61 nmol/L). AG013736 was also a potent inhibitor of CSF-1R in both assays (enzyme, IC50 = 16 nmol/L; cellular, IC50 = 21 nmol/L), whereas BAY 43-9006 is less potent in the enzyme assay (IC50 = 107 nmol/L) than in the cellular system (IC50 = 20 nmol/L). In contrast, we found that CHIR258 had less activity in the cellular assay (IC50 = 535 nmol/L) relative to its enzymatic potency (IC50 = 26 nmol/L). These results show the use of a cell-based assay to confirm the inhibitory activity of lead compounds and drug candidates, such as ABT-869, against the CSF-1R protein in situ.     

血小板功能检测的研究进展

血小板在执行生理性止血的同时,也在病理性血栓形成过程中起重要作用。血小板功能检测对于临床相关疾病的诊断和抗血小板药物的筛选及相关研究有着重要的意义。目前,血小板功能检测的实验与方法日益增多,但所有这些检测方法均有不足之处,因而有必要研究和发展一种操作简便、检测灵敏度高的方法。本文对近年来血小板功能检测方法诸如血小板的一般功能检测、血小板黏附功能测定、血小板聚集功能测定、血小板释放功能测定、血小板凝血活性检测和流式细胞术在血小板功能检测中的应用等研究进展进行了综述,并对研究前景作了简单的展望。