Barbara G. Campling

Chemosensitivity testing of small cell lung cancer using the MTT assay

A simple colorimetric test, the MTT assay, has been adapted for chemosensitivity testing of human small cell lung cancer cell lines, and fresh tumour samples. Optimal conditions for clinical chemosensitivity testing were determined using established SCLC lines. Nineteen different chemotherapeutic agents were tested, and sixteen of them were found to be cytotoxic in this assay system. The drug sensitivity of a panel of 16 SCLC cell lines was measured and compared. There was very little intraexperiment variation, but the interexperiment variation was significant. Cell lines which were derived from patients who had not received chemotherapy at the time the cell line was established were more sensitive (to all but one of the drugs) than lines derived from treated patients, and the differences were statistically significant for two of the drugs. One cell line, NCI-H209, which was derived from an untreated patient, stood out as being the most sensitive or among the most sensitive to all of the drugs tested. Another cell line, H69AR, which is a multidrug resistant subline of the cell line NCI-H69, was the most resistant to many of the natural product drugs tested. Multiple drug chemosensitivity testing was performed on eight fresh tumour samples from SCLC patients (five pleural effusions, one lymph node, and two primary tumours). It was possible to perform chemosensitivity testing on all of the clinical samples in which sufficient tumour cells were available. The drug sensitivity of the clinical samples was, in most cases, within the same range as for the cell lines. Since this assay is very rapid and simple to perform, it may have practical applications in clinical drug sensitivity testing of human tumours.

Use of the mtt assay for rapid determination of chemosensitivity of human leukemic blast cells

A microcytotoxicity assay employing a tetrazolium salt has been adapted for testing the response of human leukemic blast cells to a variety of chemotherapeutic agents. After exposure to various concentrations of drugs, the viability of fresh leukemic blast cells was measured using a tetrazolium salt, MTT, which is converted to blue formazan crystals by living cells. The amount of formazan produced was quantitated using a microtitre plate spectrophotometer. In the present study, optimal conditions for chemosensitivity testing of human leukemia samples were determined, and the relative chemosensitivity of five patient samples was tested.

Human monoclonal antibodies.

SummaryThe technology for the production of murine monoclonal antibodies has been refined enormously since its introduction in 1975. However, the technology for generating human monoclonal antibodies has only recently come into its own. In this review, three currently available approaches to the production of human monoclonal antibodies are described. These include the hybridoma technique, based on the fusion of antibody-producing human B lymphocytes with either mouse or human myeloma or lymphoblastoid cells; the EBV immortalization technique, based on the use of Epstein-Barr virus (EBV) to ‘immortalize’ antigen-specific human B lymphocytes; and the EBV-hybridoma technique, based on a combination of the first two methods.The EBV-hybridoma system retains the advantageous features of the other two systems while overcoming their pitfalls and may be the current method of choice for producing human monoclonal antibodies with a defined specificity.

The HDAC inhibitor panobinostat (LBH589) inhibits mesothelioma and lung cancer cells in vitro and in vivo with particular efficacy for small cell lung cancer

Lung cancer is the leading cause of cancer deaths in the United States. Current therapies are inadequate. Histone deacetylase inhibitors (HDACi) are a recently developed class of anticancer agents that cause increased acetylation of core histones and nonhistone proteins leading to modulation of gene expression and protein activity involved in cancer cell growth and survival pathways. We examined the efficacy of the HDACi panobinostat (LBH589) in a wide range of lung cancers and mesotheliomas. Panobinostat was cytotoxic in almost all 37 cancer cell lines tested. IC50 and LD50 values were in the low nmol/L range (4–470 nmol/L; median, 20 nmol/L). Small cell lung cancer (SCLC) cell lines were among the most sensitive lines, with LD50 values consistently <25 nmol/L. In lung cancer and mesothelioma animal models, panobinostat significantly decreased tumor growth by an average of 62% when compared with vehicle control. Panobinostat was equally effective in immunocompetent and severe combined immunodeficiency mice, indicating that the inhibition of tumor growth by panobinostat was not due to direct immunologic effects. Panobinostat was, however, particularly effective in SCLC xenografts, and the addition of the chemotherapy agent etoposide augmented antitumor effects. Protein analysis of treated tumor biopsies revealed elevated amounts of cell cycle regulators such as p21 and proapoptosis factors, such as caspase 3 and 7 and cleaved poly[ADP-ribose] polymerase, coupled with decreased levels of antiapoptotic factors such as Bcl-2 and Bcl-XL. These studies together suggest that panobinostat may be a useful adjunct in the treatment of thoracic malignancies, especially SCLC. [Mol Cancer Ther 2009;8(8):2221–31]

Enhanced anti-cancer activities of some derivatives of titanocene dichloride

The compounds (eta5-C5H5)2TiCl2 (I), currently undergoing phase II trials, (eta5-C5H5)(eta5-C5H4CO2Me)TiCl2 (II) and C5H4CO2Me)2TiCl2 (III) are assessed for their efficacies against a small lung cancer cell line. It is found that the introduction of the electron withdrawing carbomethoxy group into the cyclopentadienyl rings increases the effectiveness of this class of drugs, such that III compares favorably with the well known cisplatin.

Secretion of atrial natriuretic peptide and vasopressin by small cell lung cancer

Background. Hyponatremia in patients with small cell lung cancer (SCLC) is a common clinical problem usually attributed to tumor secretion of arginine vasopressin (AVP). It recently was shown that some SCLC cell lines produce atrial natriuretic peptide (ANP). The purpose of this investigation was to determine the frequency and clinical consequences of secretion of ANP by SCLC and the relative contribution of ANP and AVP to the hyponatremia associated with this disease.

Clinical implication of p53 mutation in lung cancer

Lung cancer development involves multiple genetic abnormalities leading to malignant transformation of the bronchial epithelial cells, followed by invasion and metastasis. One of the most common changes is mutation of the p53 tumor suppressor gene. The frequency of p53 alterations in lung cancer is highest in small cell and squamous cell carcinomas. A genetic “signature” of the type of p53 mutations has been associated with carcinogens in cigarette smoke. The majority of clinical studies suggest that lung cancers with p53 alterations carry a worse prognosis, and may be relatively more resistant to chemotherapy and radiation. An understanding of the role of p53 in human lung cancer may lead to more rational targeted approaches for treating this disease. P53 gene replacement is currently under clinical investigation but clearly more effective means of gene deliver to the tumor cells are required. Novel approaches to lung cancer therapy are needed to improve the observed poor patient survival despite current therapies.

Mechanisms of Resistance of Human Small Cell Lung Cancer Lines Selected in VP-16 and Cisplatin

The combination of VP‐16 and cisplatin is one of the most active regimens available for the treatment of small cell lung cancer (SCLC), however, most tumors eventually become resistant to these drugs.

Constitutive activation of met kinase in non‐small‐cell lung carcinomas correlates with anchorage‐independent cell survival

Lung cancer is currently the most frequent cause of cancer death in North America. Hepatocyte growth factor (HGF) and its receptor Met are frequently over‐expressed in non‐small‐cell lung carcinomas (NSCLC), but their potential role in tumor progression is not clearly known. To assess the role of HGF/Met signaling in lung carcinomas, we have examined the expression, activation status, and function of Met in NSCLC cell lines (n = 7), established from primary tumors or pleural fluids of cancer patients. We observed Met expression in three NSCLC cell lines, two of which exhibited constitutive tyrosine‐phosphorylation of Met, and Met kinase activity. In addition, the observed constitutive activation of Met was sustained under anchorage‐independent conditions, and correlated with phosphatidyl inositol 3‐kinase‐dependent cell survival. Immunoreactive HGF‐like protein was secreted by two Met‐positive and two Met‐negative NSCLC cell lines. However HGF activity, as determined by the ability to induce cell scattering and tyrosine‐phosphorylation of Met in reporter cell lines, was detected in conditioned medium from only one Met‐negative NSCLC cell line: none of the conditioned media from Met‐expressing NSCLC cell lines showed detectable HGF activity. Thus, constitutive activation of Met in NSCLC cell lines may occur at least in part through intracrine, or HGF‐independent mechanisms. Interestingly, additional paracrine stimulation with exogenous recombinant HGF was required for DNA synthesis and correlated with increased activation of ERK1/2 in all Met‐positive NSCLC cell lines, regardless of the basal activation status of Met. These findings indicate that a medium level of constitutive activation of Met occurs in some NSCLC cell lines, and correlates with survival of detached carcinoma cells; whereas additional paracrine stimulation by recombinant HGF is required for DNA synthesis. Thus constitutive and paracrine activation of Met may provide complementary signals that promote survival and proliferation, respectively, during tumor progression of NSCLC. J. Cell. Biochem. 86: 665–677, 2002.

Establishment of a human large cell lung tumor line (QU‐DB) with metastatic properties in athymic mice

A continuous human cell line was established from a patient with large cell anaplastic lung carcinoma. This cell line, designated QU‐DB, has been in culture for over 36 months and grows as an adherent monolayer with a doubling time of 10–12 hours. Its morphology, ultrastructure, karyotype, ability to grow in soft agar and heterotransplantability, indicate it is a large‐cell lung tumor cell line of human origin. Three cell lines were established from metastatic tumors in nude mice receiving subcutaneous injections of QU‐DB cells. The morphology and growth characteristics exhibited by these cell lines were similar to the primary cell line. Karyotypic analysis of cell lines derived from the primary tumor and a metastasis to the diaphragm were similar, but cells from a liver metastasis culture showed additional karyotypic changes. This large cell lung tumor cell line may prove useful as a model system for studies of human tumor progression and metastasis.