Chapter 3 Effect of antimalarial drugs, haemozoin ( β -haematin), Plasmodium
3.3 Results
3.3.4 Analysis of the relative expression of GTP-cyclohydrolase 1 mRNA
In summary, the anti-neopterin IgY produced in chickens immunised with a neopterin-rabbit albumin conjugate recognized neopterin-ovalbumin conjugates and neopterin added to glutaraldehyde-activated wells when they were used as coatings in an indirect ELISA. However, the antibodies did not titrate in a competitive ELISA.
3.3.4 Analysis of the relative expression of GTP-cyclohydrolase 1 mRNA
Figure 3.10. Agarose gel (1% w/v) of quantitative PCR products amplified from cDNA generated from U937 RNA, using primers for ββββ-actin and GTP-cyclohydrolase 1 (Primer set A and B). Using reverse transcriptase, cDNA was synthesized from total RNA isolated from U937 cells. RNA was either treated with DNase (Lanes 4, 5, 8, 9, 12 and 13) or not (Lanes 6, 7, 10, 11, 14 and 15). cDNA was amplified using SYBR® Green quantitative PCR, an annealing temperature of 55ºC and primers for β-actin (Lanes 4, 5, 6 and 7), GTP-cyclohydrolase 1 primer set B (Lanes 8, 9, 10 and 11) and GTP-cyclohydrolase 1 primer set A (Lanes 12, 13, 14 and 15). The quantitative PCR products were run on a 1% (w/v) agarose gel in the presence of ethidium bromide. Lane 1: Fermentas MassRuler™ DNA Ladder Mix (U.S.A.), Lane 2, no- template control for GTP-cyclohydrolase 1 primer set B, lane 3: no-template control for β-actin.
For the 2−∆∆CT method to be valid, the amplification efficiencies of the target (GTP cyclohydrolase 1 (GTP-CH1) and reference (β-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) must be comparable. To determine if the efficiencies are equal, variation in ∆CT (CT, target - CT, reference) with template dilution was monitored. The ∆CT values were plotted versus the logarithm of the cDNA input, the dynamic range of the assay was ascertained and the data were fit using least-squares linear regression analysis. The absolute values of both slopes were less than 0.1 (Figure 3.11), indicating that the amplification efficiencies of the target, GTP-CH1, and references, β-actin or GAPDH, were similar, and the
CT
∆∆
2− method could be used for relative quantification.
A
y = -0.0954x + 16.405 R2 = 0.7604
16 16.2 16.4 16.6 16.8 17 17.2
-3.3 -3.1 -2.9 -2.7 -2.5 -2.3 -2.1 -1.9 -1.7 -1.5
Log (cDNA input)
∆∆∆∆CT
B
y = -0.0967x + 7.7849 R2 = 0.9497
7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8
-3.5 -3 -2.5 -2 -1.5 -1
Log (cDNA input)
∆∆∆∆CT
Figure 3.11. Validation of the 2−∆∆CT method used to determine the relative expression of the target gene mRNA (GTP-cyclohydrolase 1) to the expression of the internal control gene mRNA (ββββ-actin OR glyceraldehyde-3-phosphate dehydrogenase). Using reverse transcriptase, cDNA was synthesized from total RNA isolated from U937 cells. Serial dilutions of the cDNA were amplified by SYBR® Green quantitative PCR using primers specific for GTP- cyclohydrolase 1 and (A) β-actin or (B) glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
The ∆CT (CT,GTP-CH1 – CT,β-actin OR GAPDH) was calculated for each cDNA dilution and plotted against the logarithm of the cDNA input. The data were fit using least-squares linear regression analysis and the slope of the trendline was determined. Results are expressed as the mean of three amplifications ± SD.
3.3.4.1 Effect of phorbol-12-myristate-13-acetate and interferon-γγγγ on the expression of GTP-cyclohydrolase 1 mRNA
Figure 3.12 demonstrates that treatment of the suspended U937 cells (Control) with phorbol- 12-myristate-13-acetate (PMA) for 48 hours produced a 2-fold increase in the expression of GTP-cyclohydrolase 1 (GTP-CH1) mRNA (p<0.05), and the addition of interferon-γ (IFN-γ) for 18 hours promoted ~15-fold increase in GTP-CH1 mRNA expression (p<0.05), both relative to the expression of β-actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. There was no significant difference (p>0.05) between the fold-change observed in the GTP-CH1 mRNA expression between the two house-keeping reference genes, β-actin and GAPDH. The β-actin house-keeping gene was used as the reference gene for further experiments.
0 2 4 6 8 10 12 14 16 18 20
Control PMA PMA & IFN-γ
Fold change in GTP-cyclohydrolase 1 mRNA expression relative toβ-actin or GAPDH
β-actin GAPDH
Figure 3.12. Effect of phorbol-12-myristate-13-acetate (PMA) alone and PMA with interferon-γγγγ (IFN-γγγγ) on the expression of GTP-cyclohydrolase 1 mRNA in U937 cells. U937 cells in suspension were used as a control. U937 cells were differentiated with 10 ng/ml phorbol-12-myristate-13-acetate (PMA) for 48 hours. The cells were then treated or not with interferon-γ (IFN-γ, 250 U/ml) for 18 hours. Total RNA was isolated and quantitative RT-PCR was performed to establish the expression of GTP-cyclohydrolase 1 mRNA relative to the expression of β-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA using the 2−∆∆CT method. Results are the representation of one of two experiments performed in triplicate in quantitative PCR ± SD.
3.3.4.2 Effect of antimalarial drugs on the expression of GTP-cyclohydrolase 1 mRNA The effects of 18 hours treatment with antimalarial drugs in addition to phorbol-12-myristate-13- acetate (PMA) and interferon-γ (IFN-γ) on the relative expression of GTP-cyclohydrolase 1 (GTP-CH1) mRNA in U937 cells were investigated. When compared to U937 cells treated with PMA and IFN-γ without drugs (control), quinine, primaquine and artemisinin significantly down- regulated the relative expression of the GTP-CH1 mRNA by 1.7-, 1.4- and 1.3-fold, respectively (p<0.05). Doxycycline also down-regulated the expression of GTP-CH1 mRNA by 1.1-fold, and amodiaquine up-regulated the mRNA expression by 1.1 fold, but both effects were insignificant (p>0.05). Chloroquine and pyrimethamine had minimal effects on the GTP-CH1 mRNA expression.
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Control (PMA &
IFN-γ)
Amodiaquine Artemisinin Chloroquine Doxycycline Primaquine Pyrimethamine Quinine
Antimalarial drugs Fold change in GTP-cyclohydrolase 1 mRNA expression
Figure 3.13. Effect of antimalarial drugs on GTP-cyclohydrolase 1 mRNA expression in U937 cells. U937 cells were differentiated with 10 ng/ml phorbol-12-myristate-13-acetate (PMA) for 48 hours. The cells were treated with IFN-γ (250 U/ml) and therapeutic concentrations of seven antimalarial drugs for 18 hours. Total RNA was isolated from the cells and quantitative RT-PCR was performed to establish the expression of GTP-cyclohydrolase 1 mRNA relative to the expression of β-actin mRNA using the 2−∆∆CT method. Results are the representation of one of two experiments performed in triplicate in quantitative PCR ± SD.
3.3.4.3 Effects of ββββ-haematin, latex beads, non-infected and P. falciparum-infected red blood cell lysates on GTP-cyclohydrolase 1 mRNA expression
The effects of β-haematin, latex beads, non-infected and P. falciparum-infected red blood cell lysate on the expression of GTP-cyclohydrolase 1 (GTP-CH1) mRNA expression were compared to the GTP-CH1 mRNA expression in U937 cells treated with PMA and IFN-γ only (control). β-haematin significantly up-regulated the relative expression of GTP-CH1 mRNA by 1.2-fold (p<0.05) and 20 µg/ml protein of P. falciparum-infected red blood cell lysate (10%
parasitaemia) significantly down-regulated the GTP-CH1 mRNA expression by 1.5-fold (p<0.05). Latex beads, non-infected red blood cell lysate (20 and 10 µg/ml) and P. falciparum- infected red blood cell lysate (10 µg/ml) had no significant effects on the expression of GTP- CH1 mRNA (p>0.05).
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Control (PMA & IFN-γ)
β-haematin 25 µg/ml
Latex Beads 0.1% (w/v)
RBC lysate 20 µg/ml
RBC lysate 10 µg/ml
Pf RBC lysate 20 µg/ml
Pf RBC lysate 10 µg/ml
Fold change in GTP-cyclohydrolase 1 mRNA expression
Figure 3.14. Effect of ββββ-haematin, latex beads, non-infected and P. falciparum-infected red blood cell lysates on GTP-cyclohydrolase 1 mRNA expression in U937 cells. U937 cells were differentiated with 10 ng/ml phorbol-12-myristate-13-acetate (PMA) for 48 hours. The cells were then treated with interferon-γ (IFN-γ) (250 U/ml) and β-haematin or latex beads or non-infected red blood cell lysate (RBC lysate) or P. falciparum-infected red blood cell lysate (Pf RBC lysate) for 18 hours. Total RNA was isolated from the cells and quantitative RT-PCR was performed to establish the expression of GTP-cyclohydrolase 1 mRNA relative to the expression of β-actin mRNA using the 2−∆∆CT method. Results are the representation of one of two experiments performed in triplicate in quantitative PCR ± SD.
In summary, GTP cyclohydrolase 1 (GTP-CH1) mRNA expression in U937 cells was up- regulated 15-fold by interferon-γ (IFN-γ) treatment. Quinine, primaquine and artemisinin down- regulated this increase in GTP-CH1 mRNA expression by 1.7- , 1.4- and 1.3-fold, respectively.
Amodiaquine, chloroquine, doxycycline and pyrimethamine had no significant effects on the GTP-CH1 mRNA expression. β-haematin up-regulated the IFN-γ-induced GTP-CH1 mRNA expression by 1.2 fold, whereas 20 µg/ml P. falciparum-infected red blood cell lysate down- regulated this induced expression by 1.5-fold. Non-infected red blood cell lysate had no significant effects on the IFN-γ-induced GTP-CH1 mRNA expression.