Drug sensitivity assays were performed using a variation of a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay on V79, CHO-AuxB1, CHRC5, NCI-H460, and NCI-H249 cell lines following optimization of experimental conditions for each cell line. Results from this assay were compared with data assimilated simultaneously by clonogenic assay and by dye exclusion assay. Good correlation was observed using the CHO-AuxB1 cell line and the pleiotropic drug-resistant mutant CHRC5, with similar degrees of relative resistance observed with both the MTT and clonogenic assays. Good correlation was observed between the clonogenic and MTT assays for 1-h drug exposures, although the MTT assay was more sensitive to vinblastine. In general, the clonogenic assay was more sensitive when continuous drug exposures were utilized, although this was primarily related to the increased drug exposure time. While the use of the MTT assay in drug sensitivity testing of primary tumor samples is limited, since contaminating normal cells may also reduce the tetrazolium, the MTT assay can be semiautomated, and therefore it offers a valid, simple method of assessing chemosensitivity in established cell lines.

https://cancerres.aacrjournals.org/content/canres/47/4/936.full.pdf

Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing.

For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.

https://cancerres.aacrjournals.org/content/canres/48/3/589.full.pdf

Feasibility of Drug Screening with Panels of Human Tumor Cell Lines Using a Microculture Tetrazolium Assay

The polyamine-biosynthetic pathway represents an inviting target for the development of agents inhibiting carcinogenesis and tumor growth. Polyamines play an essential role in the proliferation and development of mammalian cells. Deranged polyamine metabolism may be an important factor in carcinogenesis. Depletion of polyamines inhibits growth of neoplastic cells in vitro and in animal models. Several different classes of other anticancer agents may under some conditions exert enhanced effects when polyamine levels are depleted. Some suitable inhibitors of polyamine production are currently available and other promising compounds are presently being tested. It should soon prove possible to block polyamine biosynthesis at every step in the pathway. The use of these inhibitors alone and combined either with each other or with other antitumor agents will enable a full examination of the potential of this approach.

https://cancerres.aacrjournals.org/content/canres/48/4/759.full.pdf

Polyamine Metabolism and Its Importance in Neoplastic Growth and as a Target for Chemotherapy

The autocrine hypothesis proposes that a cell produces and secretes a hormone-like substance that can interact with specific membrane receptors on its surface to induce effects such as proliferation. Thus, a cancer cell could act to stimulate its own growth. Bombesin and bombesin-like peptides (BLPs) such as gastrin-releasing peptide (GRP) cause various physiological responses in mammals, including stimulation of proliferation of 3T3 mouse fibroblasts and normal human bronchial epithelial cells in vitro and induction of gastrin cell hyperplasia and increased pancreatic DNA content in vivo in rats. Human small-cell lung cancer (SCLC) cell lines produce and secrete BLPs and can express a single class of high-affinity receptors for BLPs. Exogenously added BLPs can also stimulate the clonal growth and DNA synthesis of SCLC in vitro. These findings suggest that BLPs function as autocrine growth factors for this tumour. One way to test this hypothesis is to interrupt the function of the endogenously produced BLPs. Here, we demonstrate that a monoclonal antibody to bombesin binds to the C-terminal region of BLPs, blocks the binding of the hormone to cellular receptors and inhibits the clonal growth of SCLC in vitro and the growth of SCLC xenografts in vivo. These results demonstrate that BLPs can function as autocrine growth factors for human SCLC.

Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer.

Allele loss is a hallmark of chromosome regions harboring recessive oncogenes. Lung cancer frequently demonstrates loss of heterozygosity on 17p. Recent evidence suggests that the p53 gene located on 17p13 has many features of such an antioncogene. The p53 gene was frequently mutated or inactivated in all types of human lung cancer. The genetic abnormalities of p53 include gross changes such as homozygous deletions and abnormally sized messenger RNAs along with a variety of point or small mutations, which map to the p53 open reading frame and change amino acid sequence in a region highly conserved between mouse and man. In addition, very low or absent expression of p53 messenger RNA in lung cancer cell lines compared to normal lung was seen. These findings, coupled with the previous demonstration of 17p allele loss in lung cancer, strongly implicate p53 as an anti-oncogene whose disruption is involved in the pathogenesis of human lung cancer.

https://science.sciencemag.org/content/sci/246/4929/491.full.pdf

p53: a frequent target for genetic abnormalities in lung cancer

Using a chemically defined medium containing hydrocortisone, insulin, transferrin, 17 beta-estradiol and selenium, with or without serum supplementation (2.5% v/v), continuous cell lines can be established from 72% of all fresh biopsy specimens of small cell lung cancer (SCLC) containing tumor cells. No differences were observed in the rate of establishing cell lines from newly diagnosed untreated patients, or from patients who have relapsed from prior therapy, or from a variety of different organ sites. Biochemical characterization of 50 SCLC cell lines for the expression of L-dopa decarboxylase; bombesin-like immunoreactivity; neuron-specific enolase, and the brain isozyme of creatine kinase, revealed that SCLC cell lines can be subdivided into two distinct classes: classic SCLC cell lines (35 lines), which express elevated levels of all four biomarkers; and variant SCLC cell lines (15 lines) which have undetectable levels of L-dopa-decarboxylase and bombesin-like immunoreactivity, but continue to express neuron-specific enolase and the brain isozyme of creatine kinase. The presence of the latter two markers distinguishes variant lines fron non-SCLC cell lines. In addition, four distinct classes were identified morphologically. The biomedical differences among established SCLC cell lines may account for the differences in response rates to cytotoxic therapy observed in newly diagnosed SCLC patients. A prospective study of biomarker characterization of SCLC tumors will determine if clinical differences exist between classic and variant SCLC tumors.

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Establishment and Identification of Small Cell Lung Cancer Cell Lines Having Classic and Variant Features

Production and secretion of neuroendocrine peptides by small cell lung cancer (SCLC) has been detected in the past years. Most recently the role of bombesin as an autocrine/paracrine growth modifier has been demonstrated. We used the soft agarose clonogenic assay to evaluate the influence of other neuroendocrine peptides on the in vitro proliferation of SCLC cell lines. Neuroendocrine peptides tested were adrenocorticotropic hormone, arginine vasopressin, calcitonin, glucagon, kassinin, neurotensin, physalaemin, somatostatin, and substance P. Experiments were carried out in serum-free and serum-supplemented media with and without serum-free incubation periods. Our results indicated that the amphibian undecapeptide physalaemin inhibits the clonal and mass culture growth of SCLC cell lines at picomolar concentrations. All other neuroendocrine peptides failed to influence SCLC growth in the test systems used. These results suggest a growth regulating effect of physalaemin and a potential new form of neuroendocrine peptide therapy for SCLC.

https://cancerres.aacrjournals.org/content/canres/47/9/2371.full.pdf

In vitro growth inhibition of human small cell lung cancer by physalaemin.

The effect of human recombinant leukocyte interferon A (IFN-alpha A) and DL-alpha-difluoromethylornithine (DFMO) as single drugs and in combination on the in vitro growth, cell cycle distribution, activity of the enzyme L-dopa decarboxylase, and expression of the c-myc and N-myc oncogenes was studied in human lung cancer cell lines. In vitro growth activities were tested in concentrations ranging from 10 to 50,000 IU/ml for IFN-alpha A and from 0.1 to 10 mM for DFMO by means of the soft agarose clonogenic assay using continuous drug exposure. Ten well established small cell lung cancer (SCLC) cell lines including five cell lines of the classic and five of the variant phenotype, two cell lines derived from adenocarcinoma of the lung, and one large cell lung cancer cell line were included in the study. We found that IFN-alpha A inhibited the growth only of the variant phenotype of SCLC with an approximate drug concentration yielding a 50% inhibition of colony growth of 1000 IU/ml. None of the SCLC classic cell lines was inhibited significantly. The growth inhibition of IFN-alpha A correlated with the proliferation rate of the tumor. IFN-alpha A inhibited one of two adenocarcinoma cell lines and 0 of 1 large cell lung cancer cell line. DFMO inhibited the colony formation of 10 of 10 SCLC cell lines, 2 of 2 adenocarcinoma cell lines, and 0 of 1 large cell lung cancer cell line with a drug concentration yielding a 50% inhibition of colony growth of 1 mM. No difference between the classic and variant phenotypes of SCLC was found. The combination of IFN-alpha A and DFMO resulted in an additive cytostatic effect in all cell lines tested. The same result, i.e., an additive cytostatic effect, was obtained for two SCLC cell lines that were tested in liquid culture. Neither single drugs nor their combination led to an accumulation of cells in a particular phase of the cell cycle nor did it affect the activity of the SCLC classic marker enzyme L-dopa decarboxylase. In addition, IFN-alpha A, DFMO, and their combination did not affect the expression of the c-myc and N-myc oncogenes in cell lines NCI-N417 and NCI-H526, respectively, following 4, 24, and 72 h of continuous drug exposure.(ABSTRACT TRUNCATED AT 400 WORDS)

/pdf/additive-and-differential-biological-activity-of-a-rgmtgx85x6.pdf

Additive and Differential Biological Activity of α-Interferon A, Difluoromethylornithine, and Their Combination on Established Human Lung Cancer Cell Lines

Gerold Bepler

Papers

Establishment and Identification of Small Cell Lung Cancer Cell Lines Having Classic and Variant Features

In vitro growth inhibition of human small cell lung cancer by physalaemin.

Additive and Differential Biological Activity of α-Interferon A, Difluoromethylornithine, and Their Combination on Established Human Lung Cancer Cell Lines