Cancer Biology Experimental Design Report
Cell deaths occur by necrosis, apoptosis, or autophagy. Apoptosis or programmed cell death occurs as a normal and controlled part of an organism’s growth and development, which may be pathological or physiological. Physiological apoptosis halts the proliferation of cells to facilitate the formation of organs. Apoptosis shows traits such as altered cell membrane permeability, condensation of chromatin, blebbing of the cell membrane, cell fragmentation, and DNA laddering. It is of the essence to note that cancer is the uncontrolled and abnormal growth of cells. Some anti-carcinogenic agents conduct apoptosis to avoid the uncontrolled growth of tumours. Some oncogenes suppress apoptosis while others enhance it, thus, showing that it is possible to link cancer biology with its treatment mechanisms (Ivanova et al., 2017). Therefore, apoptosis is of the essence in cancer biology and genetics since it enables the understanding of the impacts of apoptosis to provide cancer therapeutic benefits. There is need to exploit the role of apoptosis in cancer therapy through experimental means, which would provide underlying mechanisms of cancer therapy by apoptosis.
Determination of human promyelocytic (HL-60) and B-cell leukaemia or lymphoma (BCL-2) is important in determining the role of apoptosis in cancer biology. Research indicates that HL-60 and BCL-2, which are immortal, are transformed cells derived from human promyelocytic leukaemia. Besides, the BCL-2 family of proteins is crucial to the apoptotic response (Chachad et al., 2016). According to Ivanova (2017), the structurally related proteins are composed of pro-apoptotic and anti-apoptotic members that interrelate. Tumour cells can also become dependent on BCL-2 to survive in a similar manner to oncogene addiction. The HCL-2 cells are maintained in cell cultures, whereby two cells are transfected with a plasmid that produces a high level of BCL-2 gene. It blocks apoptosis in response to anti-carcinogenic drug doxorubicin (DOX) causing apoptosis in parental HL-60 cells. This makes it easy to determine the effects of BCL-2 overexpression on apoptosis. It is done by comparing the abilities of the two lines to undergo either of the two responses to anti-carcinogenic drugs, which can be qualitative (DNA laddering) and quantitative (propidium iodide assay).
Propidium Iodide Assay is based on the principle that apoptotic cells have a blebbing plasma membrane that permits entry into the cell and binds to the DNA resulting in fluorescence. It is believed that Propidium iodide is an intercalating and a fluorescent molecule, which upon mixing with nucleic acids causes fluorescent excitation and emission of visible light. The HL-60 and BCL-2 cells were treated with 1-micrometer DOX for 24 hours, and propidium iodide stain added after harvesting. The results obtained should be compared with experimental data from HL-60 and BCL-2 cells, which were not treated with DOX 1 μM before adding PI 1 μG/ml of stain and analysed on FACS machine to ascertain the validity of the results. The BCL-2 transfected proteins are pro-apoptotic since they induce pores that allow the dye to pass and combine with DNA emitting fluorescence. The Western Blot Loading Control Sampler Kit is an easily available and efficient tool that has a panel of four antibodies, which are specific to certain proteins regularly used for protein normalization. It is of the essence to note that the results could be compared with the control kit easily.
The samples were run on FACS machine, whereby the apoptotic cells showed fluorescence while normal cells failed to emit anything. The HL-60 and BCL-2 treated with doxorubicin for 24 hours showed fluorescence while the untreated culture cells showed no fluorescence. Additionally, the PI uptake that was based on the physiological state of cells and composition of cell lines may affect the staining ability. The stability of stains in storage and stock solutions, the number of cells to be stained, concentration of stains, staining incubation time, and the buffer or media used also affects the viability of results. The exposure to high light intensity can affect the interpretation of viability. Molecular techniques are important in determining whether transformed BCL-2 expresses BCL-2 proteins better than parental HL-60 cells. It is believed that HL-60 and BCL-2 transformed cells have a higher number of BCL-2 proteins than parental HL-60 cells. Molecular techniques were used to confirm the above statement.
Trypan Blue dye exclusion method is a test used to determine the number of viable cells present in a cell suspension from culture or obtained from different cell lines. It is based on the principle that live cells possess intact cell membranes that exclude some dyes like Trypan blue, Eosin among others whereas dead cells are not able to. One of the characteristics of that distinguish live cells from dead cells include the loss of transport function across the plasma membrane in dead cells. This results from loss of membrane integrity.
The results appearances of a viable cell are small, round and refractive as opposed to non-viable dead cells which appear swollen, larger, dark blue Exclusion dyes due to infiltration of the dye through the damaged cell membrane. HL-60 and BCL-2 transformed cells with more Bcl-2 proteins which induce apoptosis to have more non-viable cells than parental HL-60 cells. Thus parental cells do not become swollen and do not become coloured compared to the dead cells confirming cell death by apoptosis. There are however some limitations of this assay. Cells must be counted within 3-5 min because the number of blue-staining cells increases with time after addition of the dye. Another setback is that large numbers of samples have to be counted and this may be inconvenient to perform all the tests on a particular day by counting one cell suspension at a time before staining the next sample.
BCL-2 Protein Detection by Western Blotting is one of the important techniques used. It is also known as protein immunoblotting since an antibody is used to recognize a specific antigen (proteins). It uses SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to divide various proteins present in a given sample. Studies indicate that mitochondrion is the principal site of action of BCL-2. The separated proteins are then blotted or transferred on a cellular matrix (mitochondria-nitrocellulose or PVDF membrane) where they are stained by antibody probes specific to the BCL-2 protein. This technique involves the transfer of proteins to NC or PVDF membrane. The blocking of the unreacted sites follows to decrease the amount of non-specific protein binding, primary antibody incubation that binds specifically to BCL-2 protein, secondary antibody incubation, which an enzyme conjugate that binds to the bound primary antibody (AP and HRP), and colour detection and analysis. Designing controls for this technique were essential to guarantee accurate, reliable, and specific test results. It is of the essence to note that positive and negative controls were prepared. Nevertheless, the Western Blot Loading Control Sampler Kit is a less expensive tool that possesses a panel of four antibodies, which are specific to certain proteins used for their normalization.
The production of a colour indicates the presence of antibody-antigen complex reaction and positive test results. The quantity of colour generated is directly proportional to the level of expression of the BCL-2 protein. From the experiments, it was clear that transformed HL-60 and BCL-2 cells expressed Bcl-2 protein better due to the production of an intensified colour or visible protein bands since the transformed cells have more BCL-2 proteins that bind to anti-BCL-2 antibodies. Some of the reagents like acrylamide are toxic; hence, it is advisable to wear personal protective clothing when handling them. Chachad and colleagues (2016) showed that SDS gel and PVDF membrane are fragile; hence, the experiment requires caution and care. Another disadvantage of western blotting is time-consuming (compared to ELISA) and has a high demand regarding the experience of the experimenter. Additionally, it requires optimizing the experimental conditions like protein isolation, buffers, type of separation, and gel concentration among others.
Detection of BCL-2 Gene by PCR is the most sensitive method used to detect and quantify BCL-2 genes. Therefore, it is used to check transgenes in transformed cells. The technique is based on the principle that PCR reactions amplify DNA fragments through synthesis catalysed by heat resistant DNA polymerases upon addition of a primer (oligonucleotides) that binds to template DNA. The PCR technique uses a DNA polymerase to amplify specific target DNA sequence that generates a large amount of the genetic material for visualization. A key feature of PCR is the use of thermo-stable DNA polymerases from thermophilic organisms that can survive the high temperatures required during the PCR protocol. It also involves short DNA primers that bind to the specific target DNA sequence, triggering replication by the DNA polymerase. The BCL-2 genes are detected and amplified by agarose gel electrophoresis. The PCR process involves repeated denaturation, annealing, extension. DNA markers and size or mass ladder standard was used to determine the DNA fragment size in an agarose electrophoresis gel. The commercially available DNA ladder is used as a control where the known standard band on the commercial ladder is compared with test results. The size of the band obtained from transformed HL-60 and BCL-2 cells was larger compared to those from parental HL-60 cells due to a high number of expressed genes. Commercial DNA ladders comprising fragments of specific base pair lengths are available. These can be used for estimation of molecular size from 10bp to 12kb, which help in the easy determination of fragment size by visual reference. DNA ladders can be stained with ethidium bromide (EtBr) or SYBR safe among others. However, the method as well as the maintenance of the equipment are expensive and require technical expertise.
Caspases Activity Assay is a multi-plated and well-utilized technique to establish the occurrence of apoptosis. According to Ivanova and others (2017), Caspases are proteases that initiate and execute the programmed cell death. A unique feature of the early stages of apoptosis is the activation of Caspase enzymes, which participate in cleavage of protein substrates and subsequently disassemble the cells. Caspase assays that allow the simple detection of the active caspases in living cells. The Caspase activities are caused by cleavage of certain substances that induce the pro-apoptotic signals, including initiator caspases. The initiator caspases cleave and activate down-regulation of caspases and effector Caspases that cleave and inactivate cellular proteins leading to apoptotic responses. The assay process involves measurement of caspases-3 activity using fluorogenic substrate. HL-60 and HL-60/BCL-2 cells were provided one treated with DOX 0.4uM for 16 hours while another not treated. Upon addition of the fluorogenic substrate, a fluorimeter with an excitation filter at 390nm and emission filter at 520 was used to read the results. Positive, negative, and black controls were used to validate findings. The plates containing the cells of HL-60/BCL-2 cells with DOX showed fluorescence due to apoptosis while the normal cells did not emit fluorescence. Some commercial control kits containing reagents to measure ATP include CellTiter-Glo Luminescent Cell Viability Assay, Promega Corporation Cat. # G7570 ATPLite 1 step, Perkin Elmer Cat. # 6016731, Adenosine 5′-triphosphate (ATP) bioluminescent somatic cell assay kit among others.
In summary, apoptosis can be applied to establish the role of certain proteins or genes in cancer biology, especially in cancer therapy. Research on the role of apoptosis in cancer growth and development can be effectively used to understand the means of apoptosis and how they play a role in cancer growth and development. In this experiment the role of BCL-2 family in apoptosis was exploited. A Caspase activity is pro-apoptosis and is essential in maintaining normal physiology including tissue homeostasis. Whether a cell should die or stay alive is determined by about 20 BCL-2 protein family expression of pro-apoptotic and anti-apoptotic regulators. BCL-2 subfamily are anti-apoptosis while BCL-2 family while Bax and BH3 are pro-apoptosis. Under stress or pathological condition like neoplasm, the BCL-2 subfamily converge at intracellular membrane to determine whether the dies or not. These anti-apoptotic BCL-2 family are oncoproteins which can be activated and become pro-apoptotic. The pro-apoptotic BCL-2 then become cancer suppressors by inducing apoptosis. From this experimental results, the HL-60 and untransformed BCL-2 are anti-apoptotic. Upon activation by exposure to doxorubicin. The introduction of the DOX actives the anti-apoptotic cells, transforming them to pro-apoptotic cells capable of initiating apoptosis due to over expression of BCL-2 proteins. It is therefore a fact that expression of BCL-2 genes cause apoptosis which can supress cancer growth. This makes apoptosis an important process in research especially in the field of cancer biology.
Chachad, D., Ishizawa, J., Danter, W. R., Mills, G., Andreeff, M., & Kojima, K. (2016). The Novel Thiosemicarbazone Derivative Coti-2 Induces Mitochondrial Apoptosis in Acute Myeloid Leukemia Cells Via a p53-Independent Mechanism. Blood Journal, 128:2757.
Ivanova, H., Luyten, T., Decrock, E., Vervliet, T., Leybaert, L., Parys, J. B., & Bultynck, G. (2017). The BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as an evolutionary conserved inhibitor of IP 3 receptor channels. Cell Calcium.