Cancer research has benefitted immensely by cancer cell lines which provide a uniform sample and large number of single units. These cell lines are suitable to be studied in a variety of ways and for a variety of applications.
Essentially, cancer cell lines provide convenient representation of cancers and can be studied using several assays as can reflect a particular trait/aspect of cancer. Assays are performed for two major purposes (i) to ascertain the effect(s) of a physical or chemical agent on the cells and (ii) to understand the physiology or behaviour of cells under different conditions. Cancer research has benefitted immensely by cancer cell lines which provide a uniform sample and large number of single units.
These cell lines are suitable to be studied in a variety of ways and for a variety of applications. Essentially, cancer cell lines provide convenient representation of cancers and can be studied using several assays as can reflect a particular trait/aspect of cancer. Assays are performed for two major purposes (i) to ascertain the effect(s) of a physical or chemical agent on the cells and (ii) to understand the physiology or behaviour of cells under different conditions.
The approaches to ascertain the effects of a physical or chemical agent include testing cells for cytotoxicity, genotoxicity, proliferation, migration, invasion, and changes in signalling pathways that can affect cancer cells when exposed to a specific agent. The approaches to understand the physiology or behaviour of cells under different conditions include mechanistic studies and differential gene or protein expressions.
Typically, the assays as can be performed on cancer cells include studying individual cells or by analyzing a population of cells. Techniques ranging from simple phase-contrast microscopy to spectrophotometry, confocal microscopy and flow cytometry are now routinely performed on cultured cancer cells.
Description: Enzyme-linked immunosorbent assay kit for quantification of Mouse LIF in samples from serum, plasma, tissue homogenates and other biological fluids.
Description: Description of target: Leukemia inhibitory factor, or LIF, is an interleukin 6 class cytokine that affects cell growth by inhibiting differentiation. When LIF levels drop, the cells differentiate. The LIF was mapped gene to 22q11-q12.2 by Southern analysis of a series of mouse/human somatic cell hybrids and by in situ hybridization to the chromosomes of 2 normal males and some individuals with chromosomal rearrangements. The gene maps between the Philadelphia translocation BCR1 and the breakpoint of the translocation in cell line GM2324 at 22q12.2. LIF derives its name from its ability to induce the terminal differentiation of myeloid leukemic cells, thus preventing their continued growth. Other properties attributed to the cytokine include: the growth promotion and cell differentiation of different types of target cells, influence on bone metabolism, cachexia, neural development, embryogenesis and inflammation.;Species reactivity: Mouse;Application: ELISA;Assay info: ;Sensitivity: <10pg/ml
Description: Description of target: LIF has the capacity to induce terminal differentiation in leukemic cells. Its activities include the induction of hematopoietic differentiation in normal and myeloid leukemia cells, the induction of neuronal cell differentiation, and the stimulation of acute-phase protein synthesis in hepatocytes.;Species reactivity: Mouse;Application: ;Assay info: Assay Methodology: Quantitative Sandwich ELISA;Sensitivity: 0.039 ng/mL
Description: LIF Antibody: LIF is a pleiotropic cytokine with roles in several different systems. It is involved in the induction of hematopoietic differentiation in normal and myeloid leukemia cells, induction of neuronal cell differentiation, regulator of mesenchymal to epithelial conversion during kidney development, and may also have a role in immune tolerance at the maternal-fetal interface. LIF was initially recognized by its ability to induce terminal differentiation of myeloid leukemic cells. It is a member of the IL-6 cytokine superfamily and can be highly glycosylated. LIF signaling is transduced through the LIF-R/gp130 receptor complex, leading to the phosphorylation and activation of the JAK/STAT pathway. Recent evidence shows that LIF inhibits cardiomyogenesis in embryonic stem cells via STAT3 activation.
Description: LIF Antibody: LIF is a pleiotropic cytokine with roles in several different systems. It is involved in the induction of hematopoietic differentiation in normal and myeloid leukemia cells, induction of neuronal cell differentiation, regulator of mesenchymal to epithelial conversion during kidney development, and may also have a role in immune tolerance at the maternal-fetal interface. LIF was initially recognized by its ability to induce terminal differentiation of myeloid leukemic cells. It is a member of the IL-6 cytokine superfamily and can be highly glycosylated. LIF signaling is transduced through the LIF-R/gp130 receptor complex, leading to the phosphorylation and activation of the JAK/STAT pathway. Recent evidence shows that LIF inhibits cardiomyogenesis in embryonic stem cells via STAT3 activation.
Description: A polyclonal antibody against LIF. Recognizes LIF from Human. This antibody is Unconjugated. Tested in the following application: WB, ELISA;WB:1/500-1/2000.ELISA:1/20000
Description: A polyclonal antibody against LIF. Recognizes LIF from Human, Mouse, Rat. This antibody is Unconjugated. Tested in the following application: ELISA, IHC;ELISA:1:1000-1:10000, IHC:1:25-1:100
Description: LIF is a pleiotropic cytokine with roles in several different systems. It is involved in the induction of hematopoietic differentiation in normal and myeloid leukemia cells, induction of neuronal cell differentiation, regulator of mesenchymal to epithelial conversion during kidney development, and may also have a role in immune tolerance at the maternal-fetal interface.
Description: LIF is a pleiotropic cytokine with roles in several different systems. It is involved in the induction of hematopoietic differentiation in normal and myeloid leukemia cells, induction of neuronal cell differentiation, regulator of mesenchymal to epithelial conversion during kidney development, and may also have a role in immune tolerance at the maternal-fetal interface.
Description: LIF is a pleiotropic cytokine produced at the maternal-fetal interface which has been shown to play an essential role in implantation in mice. It is produced in high amounts by the human endometrium and the trophoblast itself, and its receptors are present on cytotrophoblast cells. LIF could thus play a role in modulating HLA-G production and immune tolerance at the maternal-fetal interface. The gene maps to 22q11-q12.2, between the Philadelphia translocation BCR gene and the breakpoint of the translocation in cell line GM2324 at 22q12.2.
Description: LIF is a pleiotropic cytokine produced at the maternal-fetal interface which has been shown to play an essential role in implantation in mice. This gene is mapped to 22q11-q12.2, between the Philadelphia translocation BCR gene and the breakpoint of the translocation in cell line GM2324 at 22q12.2. LIF is produced in high amounts by the human endometrium and the trophoblast itself, and LIF receptors are present on cytotrophoblast cells. It could, thus, play a role in modulating HLA-G production and immune tolerance at the maternal-fetal interface.
Description: Mouse LIF protein, His tag, expressed in human 293 cells. (Uniprot ID: P09056)
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Also, cells cultured in 6, 12, 24 and 96 well plates are utilized for the assays owing to the ease in which several concentrations of a variable being tested can be studied in either duplicates, triplicates or in more multiples so as to make the results meaningful and reliable.
There are parallel advancements in both the techniques being developed and in the instrumentation/equipment/software that help in data acquisition and analysis. In fact, several journals from reputed publishing houses focus only on cell-based assays and in vitro screening/testing.
Though all journals dealing with cell biology and cell physiology have specific manuscript categories such as “from the bench”, “tools and techniques” where assays and in vitro testing developments are considered, we present the journals with a specific focus in these areas in Table 1. We present in this minireview, the commonly used assays utilizing cancer cell lines and their specific applications. Also, the recent developments in cellbased assay technologies are mentioned.
Assays
Cytotoxicity
The ability of a drug to induce cell death is an integral part of chemotherapy. The assays which test for the cytotoxicity of a drug are classified as genomic or proteomic assays, functional assays, cytology assays and mitochondrial assays.
Adenosine tri phosphate (ATP) assay is a functional assay which is the quickest cell viability/cytotoxicity assay. When the cell undergoes apoptosis or necrosis, it is seen that the ATP levels decline significantly. This is used as a biomarker for cytocidal effects. The levels of ATP are quantified using luciferase enzyme which results in luminescence which is recorded.
Lactate dehydrogenase leakage assay (LDH assay) is a commonly used cytotoxicity assay in the pharmaceutical industry. When there is a loss in membrane stability of a cell induced by cell death there is release of LDH into the culture medium. The LDH levels are thus directly proportional to the amount of cell death which is detected by the addition of a tetrazolium salt.
The resazurin reduction assay is similar to MTT and ATP assay. In this technique the resazurin when added to metabolically active cells gets converted to resorufin a pink fluorescent compound. The level of resorufin is indicative of cell viability. This assay is very sensitive and the quantification of resorufin is done by recording fluorescence at 560 nm (excitation) and 590 nm (emission).
Trypan blue assay or dye exclusion assay is the most commonly used cytotoxicity/cell viability assay. The dead cells whose membrane integrity has been disrupted will stain blue, whereas the live cells which maintain their membrane integrity exclude the dye. Thus the differential staining helps in characterisation of live and dead cells which gives the data for cell viability. This is a hemocytometer based assay.
The staining of cells with propidium iodide (PI) and acridine orange (AO) results in the live cells fluorescing green and dead cells fluoresce orange when viewed under a dark field fluorescence microscope. Here the percentage inhibition is derived from the cell viability value as in the case of trypan blue assay.
Genotoxicity
Induction of damage to the genetic material of a cell either by a physical or chemical agent to is termed genotoxicity. These agents can directly or indirectly affect the DNAof somatic or germ line cells causing mutations or other aberrations that can alter normal cellular functions.
The propagation or effects of such damages are prevented by cells through a complex processes, the DNA repair and apoptosis. Various in vitro cell culture techniques are functional to examine the extent of damage caused to DNA in vivo.
These cell culture models utilize assays such as comet assay, DNA fragmentation assay, cell viability assay and various high throughput technologies for the evaluation of DNAtoxicity. Several cytogenetic techniques are used to test for genotoxicity.
These include chromosomal numerical and structural aberrations, micronucleus assay, and sister chromatid exchange assay. Comet assay is one such widely used technique to assess DNA strand break in a single cells and allowing a large number of cells to be analyzed simultaneously. This technique involves embedding of cells on agarose coated slides which is followed by cell lysis and DNA unwinding using detergent and salt respectively.
In the process of electrophoresis, the damaged nuclear material travels away from the cell which can be visualized as a tail detached from the head. DNA specific fluorescent dyes such as ethidium bromide or propidium iodide can be utilized for imaging the amount of comet tail as compared to the compactness of the head to reveal the intensity of DNA strand breaks. There are commercially available software for large-scale analysis of such comets.
Agarose gel electrophoresis technique can also be utilized for studying DNAfragmentation caused by cellular apoptosis which is triggered by caspase activated DNAase (CAD). The fragmentation of DNAinto small 200 base pair nucleotides can be visualized using fluorescent dyes.
The technique can be effectively used to evaluate chemical and radiation induced genotoxicity on cell lines. A technique which utilizes fluorescent dyes such as DAPI (4′,6- diamidino-2-phenylindole) for imaging is γH2AX assay. This technique determines the proportion of γH2AX phosphorylation in the nucleus as a response to radiation induced strand breaks.
The amount of γH2AX foci thus, has an undeviating association with the amount of radiation exposure. This technique is rapid and reliable for estimating the dose response of radiation exposure. Few other assays such as micronucleus assay and chromosomal aberration assay can be utilized for dose estimation of genotoxic agent.
Computer based analysis of genotoxicity has recently been developed for the analysis of large number of samples at a high speed and sensitivity. Quantitative structure activity relationships (QSAR) can be efficiently used to predict the relationship between genotoxicity and genotoxic efficacy of the agent used for exposure. This is a technique used for evaluation of DNA damage as a high throughput approach and for dose estimations.
Cell Migration
Drug development against cancer previously focused mainly on assays that screen cytotoxic agents which aid in inhibition of cell proliferation or stimulates apoptosis. The attention has now shifted to targeted therapies with development of inhibitors of invasion.
Invasion and metastasis are accompanied by alterations in cell migration. Commonly used in vitro cell migration assays include the Boyden chamber assay and the scratch wound healing assays. Advancement in this area is the integration of analysis systems with microfluidics.
In wound healing assay, the cells are maintained as monolayer cultures. At 80% confluency, the cells are exposed to Mitomycin-C which is an inhibitor of cell division.
One hour post incubation, a scratch (wound) is made using a sterile micropipette tip and this will be observed under phase contrast inverted microscope at various time intervals and the width of the wound will be measured.
To visualize the migration of the cells, fluorescent microscopic techniques can also be used and the documentation of the scratch can be made using time-lapse imaging. Scratch assay is used to test the effect of certain drugs on the invasiveness of cells. Its major drawbacks are the time taken to perform the assay and the absence of chemoattractants.
The Boyden chamber assay (trans-well migration assay, filter membrane migration assay or chemotaxis assay) on the other hand consumes lesser time and employs chemoattractants. In this assay, a microporous membrane is utilized which divides the culture well into two. In one well, usually the upper compartment the cells are cultured and the lower compartment contains chemotactic agents.
Usually serum is used as a chemoattractant and the cells are cultured in serum free medium. The migratory cells can be quantified using a plate reader or by staining post fixing of the membrane. Microfluidic assays have of late emerged with the potential to be physiologically relevant for testing the migratory capabilities of cells when exposed to a drug. The device offers dual ports, where one port is used to introduce cells and the other port is used to deliver the drugs.
The cells once introduced into the device attach at the bottom and the drug delivered through the port at the other end of the chamber sets up a gradient. The cells are imaged to visualize migration. Paper based invasion assay pools common invasion assays and microfluidic devices to create a gradient which is similar to those in vivo. This technique employs “cells in gels in paper” (CiGip).
The waxed papers are coated with hydrogels and seeded with cells and stacked one on top of the other to resemble tissues. The migration of the cells is then quantified.
Cell Proliferation
In vitro assays are performed to quantify proliferation which reflects cellular responses to various stimuli. These techniques help in monitoring cell division, number of cells generated over time, the proliferative ability of cells and DNA synthesis.
Cell Proliferation Essay
Traditional proliferative assays are performed in drug testing to assess the effect of drug on cell proliferation. Conventionally, cell proliferation is being assessed by its DNA synthesizing capacity. The cell lines are incubated few hours to overnight with 3Hthymidine (a radioactive label). Proliferating cells integrate the radioactive label during the process of DNA synthesis which can be visualized and counted using a scintillation counter.
A similar assay is performed to evaluate cell proliferation which utilizes 5- bromo-2′-deoxyuridine (BrdU). BrdU also gets incorporated into the newly synthesized DNA which can be measured by a BrdU specific monoclonal antibody. A secondary antibody targeting the primary BrdU monoclonal antibody acts as a reporter generating a fluorescent signal on DNA synthesis indicating proliferation.
This technique utilizes a calorimeter or a fluorescent microscope for the detection of reporter signals. MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) cell proliferation assay is widely used for measuring cell proliferation with respect to cellular metabolism.
This technique was the first high throughput assay designed for a 96 well plate format and involves a reduction of tetrazolium salts by dehydrogenase enzymes generating NADH and NADPH. The resulting metabolite being formazan (purple colour) can be measured and quantified by spectrophotometry by measuring the absorbance at 570 nm. This chemical reaction yielding a purple product is displayed only in metabolically active cells thereby eliminating non-proliferating cells.
A high throughput quantification of cell proliferation can be achieved by measuring the amount of ATP generated in the cells. This technique is based on the concept that dead cells/nonproliferating cells generate negligible amount of ATP. Enzyme luciferase generates light in the presence of ATP which can be efficiently detected by a luminometer.
This assay is efficiently used to screen large number of samples and provides a reliable readout. Another high throughput analysis of cellular proliferation utilizes a chemical reaction involving the conversion of nonfluorescent dye resazurin (blue) to a fluorescent dye resorufin (pink) in a reducing growth medium due to cell proliferation. The fluorescent signal is then measured at particular wavelengths (570 and 600 nm).
Gene expressions using mRNA
Gene expression studies on cell lines provide genetic information associated with cellular functions such as proliferation, apoptosis, cell division, adhesion and intercellular communication. Various cell lines are used for establishing gene expression profiles and genetic alterations in response to external and internal stimuli.
In vitro cell culture models help estimating genetic expressions which are effectively used in drug testing and formulation of personalized medicine. mRNA as the study material and its quantification (rather than DNA) is widely being used to understand the alterations in gene expression. This is due to the possibility of post transcriptional changes in the sequence of DNA which will be predominantly reflected in mRNA.
Quantitative Polymerase Chain Reaction (qPCR) technique has been incorporated into the laboratory routine by various researchers working on cancer cell lines. Such studies are also useful for analyzing various metabolizing genes indicating drug effects.
Gene polymorphism studies can also yield information about genetic alterations. Restricted fragment length polymorphism (RFLP) is a widely used technique to analyze nucleotide polymorphisms identified using primers. Specific primers are helpful in projecting the variations between normal and abnormal DNAsequences.
Protein estimation and detection can also predict gene expressions changes at translational levels. Western blot is a common technique used best for protein detection/identification which can be directly associated to genetic alterations. Alterations in protein levels are well established in various cell lines such as A549 and their associations with genes involved are well studied using western blot analysis and SDS PAGE (Sodium Do-decyl sulphate Poly Acrylamide Gel Electrophoresis) techniques.
High throughput analysis of gene expressions is becoming very useful in generating reliable outcomes for huge sample size and complicated gene expression profile. Microarray based primer hybridization efficiently integrates with high or low quality DNAthus generating fluorescent images. Quantification of the outcome is based on the amount of fluorescence obtained. Statistical T tests can be used to validate the microarray results.
Bioinformatics tools yield highly reliable information about well established genes and their alterations of expressions due to commonly known toxic exposures.
Protein expression
Protein expression is a critical aspect to be studied on cancer cell lines as it directly reflects the genetic characteristics of a cell. Alterations in protein expressions are noted as vital biomarkers decisive of particular disorders, especially cancer.
Several studies are being performed on various cancer cell lines for identification of biomarkers helpful in cancer diagnostic, therapeutic and prognostic applications. Western blot analysis is a well known tool for the identification of protein expression in cancer cell lines. For example, A549 cell line was efficiently utilized in a study which estimated the levels of VEGF and GAPDH proteins after drug exposures. Protein was primarily extracted from the cell line using RIPA buffer.
The extracted protein was analyzed by using SDS PAGE technique from which the proteins were hybridized to a membrane which consisted of primary antibody. After few washes, the membranes incubated with horseradish peroxidase (HRP) conjugated secondary antibodies will then be subjected to spectrophotometer for the estimation of relative optical density (ROD).
Several bioinformatics tools are available for acquiring instant information about a well studied protein. These tools are also helpful in establishing individual protein structures along with their expression levels. Uniprot, Sopma and Rasmol can be effectively utilized to study the primary, secondary and tertiary structures of proteins.
Variety of protein expression estimation software is also available to quantify the expression of isolated protein from cell lines. Nuclear Magnetic Resonance (NMR) spectroscopy and X-ray crystallography are effective techniques used for studying 3D structure of proteins at molecular level with high atomic resolution. NMR spectroscopy can generate 3D structures of protein samples with size as low as 5 to 25 KDa. Cell free expression systems are recently developed for the investigation of membrane proteins using NMR spectroscopy.
This technique is proposed to be highly beneficial as it presents amino acid type selective position and specific labelling. NMR spectroscopy has also proved effective in isotope labelling of proteins.
Estimation of histone proteins is a common technique for analyzing DNA breaks associated with radiation effects on cells. γH2AX assay explores the amount of phosphorylation of γH2AX protein in response to radiation damages. γH2AX antibodies are utilized in this technique which is allowed to integrate with γH2AX proteins in the nucleus which can then be visualized as individual foci in fluorescent microscope.
A few recent developments
Recent developments in utilizing cell lines aim at high throughput screening and protocols with simpler steps that earlier ones. For example, the latest approaches include pre-seeded 96 well plates for assays as supplied by Cell Applications, Inc., San Diego, CA 92121, USA.
Such pre-seeded cells in 96-well plates in appropriate cell type-specific medium provided for convenient material as can be used for several assays. Currently, several human and animal primary cells including adipocytes, astrocytes, cardiomyocytes, chondrocytes, endothelial cells, epithelial cells, fibroblasts, hair cells, keratinocytes, melanocytes, neurons, skeletal muscle cells. Stem cells and synoviocytes are available in this format.
Cells that are pre-screened for specific signalling activity are also being provided for a variety of applications. One recent development in the assay technology is through the GeneQuery™ qPCR arrays as supplied by Krishgen BioSystems. These arrays are convenient to study gene expressions of a variety of cells and cell lines.
The target cells include cell lines, stem cells and primary cells of human origin. Similarly, qPCR array kits in 96 well formats to study apoptosis which include 88 apoptosis-related genes are now available for rapid assays. Another convenient development is the Kinetic TM blue assay (KB assay) reagent which makes proliferation and cytotoxicity assays rapid and convenient.
This thymidine assay utilizes a single-step, overnight incubation method to monitor cell growth, viability and proliferation. This technique is a calorimetric/fluorometric assay which utilizes absorbance values to determine the end point. Simultaneous analysis of large numbers of samples calorimetrically is also possible by the TM Quantichrome cytotoxicity assay kit which utilizes detection of lactate dehydrogenase (LDH) to detect apoptosis.
Conclusion
Various Assays as can be performed on cell lines serve as valuable tools in determining certain specific aspects of the cell as summarized in Figure 1. These tools continue to yield productive outcomes which are being effectively implemented in various applications such as drug discovery and pharmacokinetics.
High throughput screening of cells is becoming popular in recent times which support handling of complex characteristics of cell with huge sample sizes. These high throughput techniques provide results of good quality with high sensitivity, reliability and reproducibility. Conventional laboratory techniques for identification of certain specific aspect of cell are gradually being replaced by rapid high throughput screening and testing procedures.
Also, with several “ready-to-use” material inputs from commercial suppliers, the field of in vitro testing and assay development is becoming more efficient and advanced.
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