We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.

https://www.researchgate.net/file.PostFileLoader.html?id=50d0b614e24a467d68000010&assetKey=AS%3A273569009864704%401442235342217

New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening

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 US National Cancer Institute (NCI) 60 human tumour cell line anticancer drug screen (NCI60) was developed in the late 1980s as an in vitro drug-discovery tool intended to supplant the use of transplantable animal tumours in anticancer drug screening. This screening model was rapidly recognized as a rich source of information about the mechanisms of growth inhibition and tumour-cell kill. Recently, its role has changed to that of a service screen supporting the cancer research community. Here I review the development, use and productivity of the screen, highlighting several outcomes that have contributed to advances in cancer chemotherapy.

The NCI60 human tumour cell line anticancer drug screen

Functional activation of growth factors and receptors of the epidermal growth factor receptor (EGFR) family occurs in most epithelial-cell cancers, rendering EGFR a target for cancer treatment. This article discusses the mechanisms of action of EGFR inhibitors, their anticancer activity, and clinical issues concerning their use in the treatment of patients with cancer.

https://www.nejm.org/doi/pdf/10.1056/NEJMra0707704

EGFR Antagonists in Cancer Treatment

To reveal the antiangiogenic capability of cancer chemotherapy, we developed an alternative antiangiogenic schedule for administration of cyclophosphamide. We show here that this antiangiogenic schedule avoided drug resistance and eradicated Lewis lung carcinoma and L1210 leukemia, an outcome not possible with the conventional schedule. When Lewis lung carcinoma and EMT-6 breast cancer were made drug resistant before therapy, the antiangiogenic schedule suppressed tumor growth 3-fold more effectively than the conventional schedule. When another angiogenesis inhibitor, TNP-470, was added to the antiangiogenic schedule of cyclophosphamide, drug-resistant Lewis lung carcinomas were eradicated. Each dose of the antiangiogenic schedule of cyclophosphamide induced the apoptosis of endothelial cells within tumors, and endothelial cell apoptosis preceded the apoptosis of drug-resistant tumor cells. This antiangiogenic effect was more pronounced in p53-null mice in which the apoptosis of p53-null endothelial cells induced by cyclophosphamide was so vigorous that drug-resistant tumors comprising 4.5% of body weight were eradicated. Thus, by using a dosing schedule of cyclophosphamide that provided more sustained apoptosis of endothelial cells within the vascular bed of a tumor, we show that a chemotherapeutic agent can more effectively control tumor growth in mice, regardless of whether the tumor cells are drug resistant.

https://cancerres.aacrjournals.org/content/canres/60/7/1878.full.pdf

Antiangiogenic Scheduling of Chemotherapy Improves Efficacy against Experimental Drug-resistant Cancer

Low-molecular-weight vascular-disrupting agents (VDAs) cause a pronounced shutdown in blood flow to solid tumours, resulting in extensive tumour-cell necrosis, while they leave the blood flow in normal tissues relatively intact. The largest group of VDAs is the tubulin-binding combretastatins, several of which are now being tested in clinical trials. DMXAA (5,6-dimethylxanthenone-4-acetic acid) - one of a structurally distinct group of drugs - is also being tested in clinical trials. A full understanding of the action of these and other VDAs will provide insights into mechanisms that control tumour blood flow and will be the basis for the development of new therapeutic drugs for targeting the established tumour vasculature for therapy.

Disrupting tumour blood vessels

Melanocytes are melanin-producing cells found in skin, hair follicles, eyes, inner ear, bones, heart and brain of humans. They arise from pluripotent neural crest cells and differentiate in response to a complex network of interacting regulatory pathways. Melanins are pigment molecules that are endogenously synthesized by melanocytes. The light absorption of melanin in skin and hair leads to photoreceptor shielding, thermoregulation, photoprotection, camouflage and display coloring. Melanins are also powerful cation chelators and may act as free radical sinks. Melanin formation is a product of complex biochemical events that starts from amino acid tyrosine and its metabolite, dopa. The types and amounts of melanin produced by melanocytes are determined genetically and are influenced by a variety of extrinsic and intrinsic factors such as hormonal changes, inflammation, age and exposure to UV light. These stimuli affect the different pathways in melanogenesis. In this review we will discuss the regulatory mechanisms involved in melanogenesis and explain how intrinsic and extrinsic factors regulate melanin production. We will also explain the regulatory roles of different proteins involved in melanogenesis.

https://www.mdpi.com/1422-0067/17/7/1144/pdf

Signaling Pathways in Melanogenesis

The antitumor agent amsacrine, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), when bound to double-stranded DNA, particularly poly(deoxyadenylicthymidylic acid), reduced the fluorescence of bound ethidium without physically displacing it from DNA. Fluorescence lifetime measurements showed that the reduction of fluorescence was not due to reduction of the lifetime of the excited state of ethidium. Rather, a proportion of the DNA-bound ethidium changed to a state where the fluorescence was highly quenched. Several other 9-anilinoacridine derivatives, and also 9-hydroxyellipticine, caused quenching of ethidium-DNA fluorescence, whereas 9-aminoacridine, proflavin, and ellipticine had no effect. Resonance energy transfer (Forster transfer) is not responsible for the effect since there is no spectral overlap between the absorption spectrum of any of the agents and the fluorescence emission spectrum of ethidium. It is suggested that quenching may occur as a result of reversible formation of electron-transfer complexes between the intercalating drug and the excited state of ethidium.

Quenching of DNA-ethidium fluorescence by amsacrine and other antitumor agents: a possible electron-transfer effect

Multiple drug resistance (multidrug resistance; MDR), a phenomenon whereby human tumours that acquire resistance to one type of therapy are found to be resistant to several other drugs that are often quite different in both structure and mode of action, has been recognised clinically for several decades. An important advance in our understanding of MDR came with the identification of P-glycoprotein and other related transporters that were expressed in some cancer cells and could recognise and catalyse the efflux of diverse anticancer drugs from cells. A second advance came from an understanding of the mechanism of programmed cell death or apoptosis, leading to MDR mediated by increased to resistance to anticancer drug-induced apoptosis. A third advance came with the finding that the proliferation of human tumours was driven by a small population of self-renewing tumour cells, focussing attention on the MDR properties of these so-called tumour stem cells rather than on the cells that comprised the majority of the tumour population. A fourth advance was the delineation of features of the tumour microenvironment, including immunosuppression, which essentially provided tumour stem cells with an MDR phenotype. Most published work on the overcoming of MDR has concentrated on inhibition of drug transporters but the complexity of mechanisms contributing demands a broad strategy for the development of methods to overcome MDR in a clinical setting.

Multiple Drug Resistance Mechanisms in Cancer

The development and progression of melanoma have been attributed to independent or combined genetic and epigenetic events There has been remarkable progress in understanding melanoma pathogenesis in terms of genetic alterations However, recent studies have revealed a complex involvement of epigenetic mechanisms in the regulation of gene expression, including methylation, chromatin modification and remodeling, and the diverse activities of non-coding RNAs The roles of gene methylation and miRNAs have been relatively well studied in melanoma, but other studies have shown that changes in chromatin status and in the differential expression of long non-coding RNAs can lead to altered regulation of key genes Taken together, they affect the functioning of signaling pathways that influence each other, intersect, and form networks in which local perturbations disturb the activity of the whole system Here, we focus on how epigenetic events intertwine with these pathways and contribute to the molecular pathogen

https://www.tandfonline.com/doi/pdf/10.1080/15592294.2014.1003746

Bruce C. Baguley

Papers

Disrupting tumour blood vessels

Signaling Pathways in Melanogenesis

Quenching of DNA-ethidium fluorescence by amsacrine and other antitumor agents: a possible electron-transfer effect

Multiple Drug Resistance Mechanisms in Cancer

Epigenetic regulation in human melanoma: past and future