Cdc20gene expression in AML cell lines
Evaluating the possible mechanism of action of a novel anti-fungal drug on cdc20gene expression in AML cell lines
Acute myeloid leukemia (AML) is fast progressing cancer of the blood which originates in the bone marrow. Cdc20 is a specific cell division checkpoint protein that helps in the activation of the anaphase-promoting complex (APC) and promotes premature mitosis leading to the fast progressive nature of cancer in AML. Increased expression of Cdc20 has been implicated in various cancerous cells and is considered to be a potential target for anti-cancer drugs.
The present study was conducted to evaluate the mechanism of action, of a novel drug used to treat AML. The drug is an established antifungal agent and has been shown to cause cytotoxicity in AML cell lines. Hence, the present study evaluated the impact of the novel drug on Cdc20 expression. The evaluation was based on RNA levels and cDNA expressions, derived from AML cell lines compared to non-cancerous cell lines.
Reverse transcription was used to get the cDNA imprints. This was essential to probe the cDNA derived from the RNA samples with the cdc20 probe. The qRT-PCR approach was implemented to evaluate the changes in expression of RNA samples (based on cDNA expressions) that were treated with the experimental drug compared to samples that were untreated. This method was used to quantify the expression of RNA over certain periods of time. A housekeeping gene GAPDH was incorporated as an endogenous control.
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The Cycle Threshold (CT) signals were detected as the positive reaction in the qRT-PCR assay. The CT levels are inversely proportional to the amount of target nucleic acid in a sample. The average CT values were highest for the sample that was treated by a drug for the cdc20 gene. This indicated the novel drug must have reduced the overall expression of the cdc20 gene compared to the samples which were untreated with the novel drug.
The time bound evaluation the 2^-ddCT values were evaluated. These values indicated the change of expression of cDNA in the same sample with respect to an endogenous control over a period of time. It was indicated that the novel drug reduced the expression of cdc20 by 63%, compared to calibrators. Moreover, the single time dCT values for test samples and calibrators complied with the results of relative expression of cdc20 gene
Acute myeloid leukemia (AML) is fast progressing cancer of the blood which originates in the bone marrow. The maturity of the bone marrow cells are prevented in AML. Instead, immature blast cells with increased capacity of mitosis are released into the blood. Around 11 billion blast cells are formed and released from the bone marrow. Thus, AML is a process whereby the hematopoietic precursor cells are transformed in such a way leading to limitless cell cycles and cell divisions. These cells do not undergo apoptosis or programmed cell death, which leads to the progression of cancer. AML is also designated by other classifications like myelocytic, granulocytic, myelogenous and non-lymphocytic leukemia. In this type of leukemia, the affected cells are RBCs, WBCs and Platelets but do not include lymphocytes. AML can affect various organs like the lymph nodes, liver, spleen and testicles (1).
The risk factors for AML includes smoking, chronic exposure to benzene (as it damages the DNA of bone marrow cells), chemotherapeutic treatment with alkylating agents and topoisomerase II inhibitors and presence of Down’s syndrome or Fanconi anemia. AML may also be potentiated by polycythemia or myelofibrosis or other forms of blood cancer. AML is the most common form of acute leukemia which affects older people. It is more prevalent in individuals >60 years of age and predominantly occurs in males. However, 15% to 25% cases of acute childhood leukemia are due to AML. The risk of AML increases to around 10 fold from the age range of 30-34 years to the age range in 65 to 69 years. During the former age range, there is a prevalence of AML in 1 out of 100,000 while in the later age range the prevalence of AML is 10 out of 100,000. Beyond the age of 70 years till the age of 85 years the prevalence is far higher (1).
Cdc20 is a specific cell division checkpoint protein which helps in the activation of the anaphase promoting complex (APC). It is the product of the cdc20 gene. For, a cell to undergo mitosis (cell division), anaphase is an important step. In this step, the sister chromatids are separated and start to move towards different poles from the equatorial plate (metaphase). This arrangement is important for cell division. Hence, cdc20 helps in normal cell division and progression of cancer cells (metastasis). Cdc20 binds to phosphorylated APC and activates it. Increased activation of APC/cyclin complex leads to inhibition of a protein called, Securin. Securin inhibits a cell from entering the Anaphase because it prevents the separation of chromatids to the opposite poles. Activation of APC thus helps a cell to enter the anaphase by overcoming the inhibition of Securin. Cdc20 also inhibits the cyclin-dependent kinase which was used to phosphorylate APC, after the mitotic phase is over. Hence, cdc20 acts as cell cycle checkpoints and regulates both mitosis and also inhibits premature mitosis (2).
Spindle assembly checkpoint (SAC) acts as a monitor to detect the proper alignment of chromosomes to the mitotic spindles before anaphase or cell division ensues. Thus, SAC acts as an inhibitor of APC-cdc20, and controls accurate mitosis. However, when chromosomal misalignment occurs, mitotic checkpoint complex (MCC) consisting of three proteins Mad2, Bub3 and BubR1 attach to APC-cdc20 and inactivate it. Thus, premature mitosis is prevented. In cases of malignancy this mechanism of inhibition is weakened which causes the cancerous cells to gain or lose genetic material. In AML, there is an abnormal expression of a protein called AML-1ETO which causes the chromosomal aberrations (3).
Truncated AML-1ETO has been demonstrated to interfere with the integrity of SAC. Hence, these cells produced repressed amounts of BubR1. Decreased BubR1 resulted in ineffective inhibition of APC-cdc20, which causes premature mitosis and progression of cancer in AML(3).Kasumi-1 cells (low BubR1 expressed, myeloblastic cells) and HL-60 cell line (AML cells showing normal BubR1 expression) were transfected with retrovirus-delivered BubR1. When induced by doxycycline the expression of Bub1 expression was increased these cells (4). Thus, it may be predicted that BUbR1 expressions will lead to inhibition of APc-cdc20 and inhibit premature mitosis. Targeting cdc-20 holds a promise for alleviating AML as inhibition of cdc20 and thus APC will prevent premature mitosis and progression of immature blasts into the blood.
The present study evaluated the possibilities of a novel drug in the treatment of AML. The drug has been already established as an effective antifungal and recent studies have indicated its preferential cytotoxicity in AML and leukemic stem cells, compared to non-cancerous hematopoietic stem cells. However, the mechanism of its cytotoxicity is unknown. Increased expression of Cdc20 has been implicated in various cancerous cells and is considered to be a potential target for anti-cancer drugs. Hence, the present study evaluated the impact of the novel drug on Cdc20 expression. The evaluation was based on RNA levels and cDNA expressions, derived from AML cell lines compared to non-cancerous cell lines.
Collection and treatment of samples
RNA was extracted from a cell line that was isolated from a patient suffering from AML. Some of these cell lines were treated with the novel drug and some cell lines were kept untreated. RT-qPCR was done to evaluate the gene expression of Cdc20.
Selection of samples for analysis
The final selection of RNA samples was based on the highest amount of RNA concentration and purity of the RNA extracted based on spectrophotometer readings at 260nm and 280nm. 3 pairs of Eppendorf tubes were taken. Each pair contained RNA from treated and untreated cell lines. 3 pairs were used for replication, and extract the most viable RNA content. Two samples, including one each from the replications were taken. The two samples respectively taken were sample “a” (derived from untreated cell line with 242.4ng/ul RNA concentration with OD260/OD280 = 1.87) and sample “d” (derived from drug treated cell line with 298.2ng/ul RNA concentration with OD260/OD280 = 1.85)
Reverse transcription for deriving cDNA from extracted and selected RNA samples
The samples “a” and “d” were subjected to RT-qPCR for generating cDNA of the respective RNAs. A reaction master mix was prepared for each of the samples in a 0.5ml Eppendorf tube (with appropriate amounts of reaction buffer and enzyme mix). The final reaction volumes were destined to be 20ul in PCR tubes. The tubes were filled with the requisite volume of sterile nuclease free water to make up the volume along with RNA samples. 3 such tubes for “a”, “d” and a control (which did not have any transcripts), were finally evaluated for getting the cDNA. Hence PCR tubes with “a” consisted of 4.13ul of RNA sample (242.4ng/ul RNA concentration) + 4.87ul sterile water + 10ul 2*RT Buffer +1ul 20*Enzyme Mix), “d” consisted of 3.35ul of RNA sample (298.2ng/ul RNA concentration) + 5.65ul sterile water + 10ul 2*RT Buffer +1ul 20*Enzyme Mix) and finally the tube with “no transcript” consisted of 9ul sterile water + 10ul 2*RT Buffer +1ul 20*Enzyme Mix. The PCR tubes were then subjected to thermocycler. Poly-T primers were used to align with the RNA sequences for obtaining cDNA. cDNA was used for obtaining the exact gene (target gene) portions to study the impact of the novel drug on Cdc20 gene.
Real Time Quantitative RT-PCR (qRT-PCR) for evaluating expression of target gene
The qRT-PCR approach was implemented to evaluate the change in expression of RNA samples that were treated with the experimental drug compared to samples that were untreated. Hence, this method helped to quantify the expression of RNA over certain periods of time. A housekeeping gene GAPDH was incorporated as an endogenous control measure, to standardize for the error (variations) in the input of nucleic acid. The cDNAs were collected from thermocycler. 5ng/ul cDNA were used as because the cDNA volumes were made to 40uL. This volume was made to use 20uL for the qRT-PCR. The RNA obtained earlier was extrapolated with cDNA final volume of 40ul. For each sample of cDNA which were subjected to qRT-PCR two reaction sets were used. In one reaction the expression of housekeeping gene was examined while in the other the gene of interest (cdc20) was examined., 2 replicates were used for the cDNA samples (treated and untreated with the novel drug) while the RNA (treated and untreated with the novel drug) and the control sample was assayed once.
The cDNA samples were probed with cdc20 and GAPDH primers separately. The RNA samples were not probed with cdc20 gene because there would be no expected complementation; however it was probed with GAPDH. Thus, each pair will be assayed for 4 reactions and hence there will be a total of 12 assays. For the qRT-PCR, 14ul of Sybrgreen PCR master mix was taken in the PCR tubes along with 5ul of cDNA, RNA or sterile nuclease-free water and 1ul primers were added, so the total volume was 20ul. The samples were run on ABI One plus Real time Machine.
The qRT-PCR assay was evaluated through detection of positive reaction from the accumulation of fluorescent signal from the cdc20 and GAPDH probes (Livak & Schmittgen 2001).
The qRT-PCR assay was evaluated through detection of positive reaction from the accumulation of fluorescent signal. The accumulation of such signal is denoted by their CT values. CT values indicate the number of cycles that are required for the fluorescent signal to cross the threshold level (when it crosses the background level) to get detected. The value is inversely proportional to the amount of targeted nucleic acid in the specific sample.
In this experiment, the CT values were calculated and expressed in different ways. Firstly, CT value of cdc20 (target gene) was compared to the CT of the GAPDH (reference gene) for both the samples (treated with the drug and untreated with the novel drug). The sample (treated with the drug) was considered the test sample, while the sample (untreated with the drug) was considered as the calibrator (Ref. Table 1). Next, the Δct of both the samples were calculated from the average CT values obtained from the cdc20gene and the GAPDH gene. It was done to find out exclusively the CT value generated from the cdc20 component with respect to nucleic acid background fluorescent variations (as indicated by GAPDH).
Table 1: The average CT value for cdc20 and GAPDH in calibrator and test sample
Average of CT for cdc20 Average CT value for GAPDH
Sample that was not treated with X drug (calibrator) 21.3540945053
Sample that was treated with X drug (test) 22.6331825256
In Table 1 the average CT value of cdc20 in calibrator and test samples are indicated. The CT value for cdc20 by itself was not enough to interpret the data because there might have been an error in handling the sample. For example if the volume pipette in one sample was more than the volume pipette for the other sample, chances of error would be higher. Therefore, the CT value of cdc20 in both sample types was normalized for the CT value of GAPDH (reference gene). Thus, the CT value for the GAPDH was subtracted from the CT value for the cdc20 for the parameter Δct (Table2). However, the above two data represented the average expressions of cdc20 in both the samples.
Sample that was not treated with X drug (calibrator) 7.74
Sample that was treated with X drug (test) 9.185
To evaluate the relative expression of cdc20 in both the samples at any given time or over a period of time 2^ (- ΔΔCT) method was used. The next step was to calibrate ΔCT of the test sample to the ΔCT of the calibrator. In other words, the ΔCT of the calibrator was subtracted from the ΔCT of the test sample (Table 3). The ΔΔCT obtained was1.445 which represented the change in expression of the cdc20 between the test and calibrator conditions, normalized for any differences in loading between the test and calibrator samples. 2^-ΔCT of the respective samples indicated the difference in CT values of cdc20 and GAPDH at any given time. More the value of 2^-ΔCT, more is the presence of the particular nucleic acid segment at any given time. Similarly, ΔΔCT denoted the difference in ΔCT samples over two time durations. 2^-ΔΔCT indicated that lesser is its value, lesser is the presence of that particular nucleic acid compared to the earlier time period, which means the expression of that portion of nucleic acid has decreased. 2^-ΔΔCT was calculated from the formula;
2^-ΔΔCT = 2^-((CT_target – CT_endo)_time2 – (CT_target – CT_endo)_time1)
2^-(ΔCT) ΔΔCT 2^(- ΔΔCT)
Calibrator 4.67765119 0 1
Test 1.718064601 1.445 0.367292158
The fold difference in expression in cdc20 between the calibrator and test sample was obtained by calculating 2^ (- ΔΔCT) (Table3). It was observed that the drug has down regulated the expression of cdc20 by 0.633 (Fig1).
Fig 2: ΔCT method generated essentially the same results as ΔΔCT, however, it tells the difference between the two conditions in absolute terms rather than the fold change.
2^- (ΔCT) for the test sample was 4.677 whereas 2^- (ΔCT) for the calibrator was 1.718. (Table3) (Fig2). This was another way for normalization against reference gene. The graph shows reduce 2^- (ΔCT) value in test sample when compared to the calibrator. This indicated that the drug was down regulated cdc20 expression in AML cells.
Acute Myeloid Leukemia is a life threatening condition manifested by rapid progression of immature hematopoietic stem cells in the blood. This leads to the fast progression of cancer due to uncontrolled cell division. Left untreated AML can affect various organs like the lymph nodes, liver, spleen and testicles through metastasis. The risk factors for AML include smoking, chronic exposure to benzene, treatment with alkylating agents and topoisomerase II inhibitors. AML is the most common form of acute leukemia which affects older people. It is more prevalent in individuals >60 years of age and predominantly occurs in males.
Cdc20, a specific cell division checkpoint protein has been associated with AML and other forms of cancer. Cdc20 helps in activation of the anaphase promoting complex (APC). This leads to mitosis and progression of cancer cells. Hence, targeting the cdc20 gene and controlling its expression holds the promise of prevention of premature mitosis. The decrease in premature mitosis would led t deceased progression of immature hematopoietic stem cells in the blood and alleviated the progression of AML. Drugs are aimed to target the expression of cdc20 gene either directly or indirectly. Doxycycline increased the uptake of BubRI, which lead to inhibition of APC-cdc20 complex. The direct actions are based on site-directed actions on the cdc20 genome.
The present study was conducted to evaluate the mechanism of action, of a novel drug used for the management of AML. The drug is an established antifungal and has shown the potential to alleviate AML. Increased expression of Cdc20 has been implicated in various cancerous cells and is considered to be a potential target for anti-cancer drugs. Hence, the present study evaluated the impact of the novel drug on Cdc20 expression. The evaluation was based on RNA levels and cDNA expressions, derived from AML cell lines compared to non-cancerous cell lines.
The RNA samples were obtained from the AML cell lines derived from patients, who were treated with the novel drug. They were compared to cell lines which were not exposed to the drug. Reverse transcription was used to get the cDNA imprints. This was essential to probe the cDNA derived from the RNA samples with the cdc20 probe. Single cycle PCR was conducted with oligo-T primers for the generation of cDNA. mRNA-based cDNA was prepared and selected for the final assay. This was done because mRNA might contain introns which are unrelated to the cdc20 functional protein. Hence, reverse transcription helped to isolate the specific target DNA portion through the cDNA approach which is related to the cdc20 gene.
The qRT-PCR approach was implemented to evaluate the changes in expression of RNA samples (based on cDNA expressions) that were treated with the experimental drug compared to samples that were untreated. This method was used to quantify the expression of RNA over certain periods of time. A housekeeping gene GAPDH was incorporated as an endogenous control. Equal volumes of cDNA, RNA and control were subjected qRT-PCR.
The Cycle Threshold (CT) signals were detected by the positive reaction in the qRT-PCR assay. The CT levels are inversely proportional to the amount of target nucleic acid in a sample. Thus, the CT values obtained in the experimental assay of qRT-PCR indicated the expressions of cdc20 gene and the GAPDH gene. By convention the CT values < 29 are indicative of strong positive reaction and also indicate abundant target nucleic acid are present in the sample. The CT values ranging from 30-39 indicate positive reactions with moderate amounts of nucleic acid segments present and the values from 38-40 indicate weak reactions with the minimal amount of target nucleic acid segments that might be an indication of infection or environmental contamination (Livak & Schmittgen, 2001).
The average CT values were highest for the sample that was treated with the drug for the cdc20 gene. This indicated the novel drug must have reduced the overall expression of the cdc20 gene compared to the samples which were untreated with the novel drug. Hence, the novel drug may have acted and targeted the cdc20 gene to reduce its overall expression. The conclusion was robust after controlling for the background fluorescence emitted by non-cdc20 genomes.
However, the main aim of the study was to evaluate the time-bound reduction in expression of cdc20 gene expressions. This was because cancer progresses very fact in a quick period of time and hence, the functional expression of cdc20 in presence and absence of drug-treated samples was vital. For the time bound evaluation, the 2^-ddCT values were evaluated. These values indicated the change of expression of cDNA in the same sample with respect to an endogenous control over a period of time. If the 2^-ddCT values are low over two time periods, it will indicate that there is the significant reduction in the expression of the cDNA transcripts (Livak & Schmittgen 2001).
On the other hand, if the 2^-ddCT values are high over two time periods, then it will indicate that there is no significant reduction in the expression of the cDNA transcripts over two time periods. From the fold difference in expression in cdc20 between the calibrator and test sample (Table 3), it is indicated that the novel drug reduced the expression of cdc20 by 63%, compared to calibrators. Moreover, the single time dCT values for test samples and calibrators complied with the results of relative expression of cdc20 gene (Table 3).
The study provided strong evidence regarding the mechanism of action of the novel drug. The drug might be applicable at very early stage of AML to reduce the expression of cdc20 gene mRNA. The limitations of this study may be attributed to the standardization of the sample or background noise while detecting the probes. However, adequate measures were taken for standardization and purity of the RNA samples extracted from the AML cell lines. Moreover, the use of GAPDH as the housekeeping gene and normalizing the CT values with respect to cdc20 gene provided adequate reliability and sensitivity to the study.
Since, the study elucidated the gene level targeting of the novel drug, future studies may be oriented towards finding the target of the drug on the expressed and translated cdc20 protein. This is because the study reflected that cdc20 gene expression is reduced in the target cells but it is not completely eliminated. Hence, there is every chance that some cdc20 functional protein will be available and may form the APC-cdc20 complex which may result in premature mitosis and lead to the progression of cancer. Therefore, some post translational modifications of cdc20 protein with regard to the action of novel drug may be studied. This would help in specifying the dose f the drug according to the stages of cancer (AML) and provide a better prognosis.
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