Changes in apoptotic gene expression induced by the DNA cross-linkers epichlorohydrin and diepoxybutane in human cell lines.

Real time quantitative reverse transcription PCR was used to monitor changes in apoptotic gene expression after treating cells with the DNA cross-linkers epichlorohydrin (ECH) and diepoxybutane (DEB). This article presents the data obtained from application of the comparative CT method to the amplification of twelve apoptotic genes in human MCF10-A cells and eight genes in HUVEC cells. Further insight regarding the significance of these data can be found in "Cross-linking by epichlorohydrin and diepoxybutane correlates with cytotoxicity and leads to apoptosis in human leukemia (HL-60) cells" (Le et al., 2018) [1].

Specifications Table

Value of the data

Previous studies of the cytotoxic mechanism of DNA cross-linkers have often used cancer cells, which are prone to apoptosis. MCF10-A and HUVEC cells are models of non-cancer cell lines.

These data provide information about changes in the expression of several genes involved in apoptosis after treating cells with the cross-linkers epichlorohydrin (ECH) or diepoxybutane (DEB), providing insight into the pathways by which cell death results.

These data suggest mechanistic differences in the modes of action of these structurally related compounds, which are likely to be related to the observed differences in cytotoxicities and carcinogenic potentials. These findings may also be useful in the design of chemotherapeutic cross-linking agents.

Data

Results from comparative CT tests [1], [2] for MCF10-A cells treated with ECH (Table 1) or DEB (Table 2) and for HUVEC cells treated with DEB (Table 3) are presented. Target genes are from a human apoptosis array. Positive ΔΔCT values indicate downregulation; negative ΔΔCT values indicate upregulation.

Table 1

Results from comparative CT tests for MCF10-A cells treated with ECH under conditions that maximize apoptosis and minimize necrosis (2.5 mM ECH for 48 h).

Target gene	ΔΔCт Mean	ΔΔCт SE	t-stat	p-value	Expression	N
BOK	1.457	0.445	3.275	0.031	−	5
DIABLO	3.606	1.020	3.535	0.038	−	4
PUMA	4.673	0.543	8.601	0.013	−	3
BIM	−2.189	0.724	−3.022	0.039	+	5
BAX	2.294	0.469	4.893	0.039	−	3
BAK1	2.840	0.095	29.759	0.001	−	3
APAF-1	6.479	0.390	16.620	0.000	−	6
CASP-9	5.859	0.397	14.764	0.000	−	6
CASP-8	2.785	0.230	12.130	0.001	−	4
CASP-2	3.949	0.670	5.893	0.027	−	3
TANK	1.088	0.335	3.245	0.031	−	5
BCL-2	5.229	0.274	19.074	0.003	−	3

Table 2

Results from comparative CT tests for MCF10-A cells treated with DEB under conditions that maximize apoptosis and minimize necrosis (2.0 mM DEB for 48 h).

Target gene	ΔΔCт Mean	ΔΔCт SE	t-stat	p-value	Expression	N
BOK	−1.334	0.225	−5.925	0.027	+	3
DIABLO	−1.121	0.104	−10.740	0.008	+	3
PUMA	5.353	1.460	3.667	0.035	−	4
BIM	−1.335	0.229	−5.821	0.028	+	3
BAX	3.185	0.140	22.686	0.000	−	5
BAK1	−1.217	0.094	−12.930	0.006	+	3
APAF-1	−2.509	0.828	−3.030	0.038	+	5
CASP-9	4.496	0.207	21.680	0.000	−	7
CASP-8	2.762	0.052	53.284	0.000	−	3
CASP-2	2.937	0.069	42.459	0.001	−	3
TANK	4.751	1.523	3.120	0.035	−	5
BCL-2	2.312	0.367	6.305	0.024	–	3

Table 3

Results from comparative CT tests for HUVEC cells treated with DEB under conditions that maximize apoptosis and minimize necrosis (0.05 mM DEB for 24 h).

Target gene	ΔΔCт Mean	ΔΔCт SE	t-stat	p-value	Expression	N
APAF-1	−2.496	2.211	−11.290	0.000	+	5
CASP-9	−4.197	6.262	−0.670	0.522	+	3
CASP-8	−5.144	6.393	−0.800	0.444	+	3
BAX	−2.325	0.345	−67.410	0.000	+	3
CASP-2	1.446	0.312	4.630	0.018	−	4
PUMA	−0.597	0.102	−5.870	0.009	+	4
CASP-3	1.771	0.808	2.190	0.060	−	4
BAK1	1.181	0.331	3.570	0.007	−	6

Experimental design, materials and methods

Drug treatment conditions were as follows: 2.0 mM DEB, 48 h and 2.5 mM ECH, 48 h for MCF10-A cells; 0.05 mM DEB, 24 h for HUVEC cells. Total RNA was purified from untreated and drug-treated cells. Reverse transcription and amplification were performed (Qiagen QuantiFast SYBR Green RT-PCR kit) with primers from the Human Apoptosis Primer Library (RealTimePrimers.com). Amplification conditions were as follows: 50 °C for 10 min, 95 °C for 5 min, 50 cycles of 95 °C for 10 s and 58 °C for 45 s in a StepOne Real-Time PCR system (Applied Biosystems).

CT values were determined for each gene of interest, as well as for an internal control (actin). ∆CT values were then calculated by subtracting the CT value for the actin control from the CT value for each gene of interest. To assess the effects of drug treatment on gene expression, the ∆∆CT value was calculated for each gene of interest by subtracting the ΔCT value for untreated cells from the ΔCT value for drug-treated cells (ΔΔCT = ΔCT treated – ΔCT untreated).

Subject area	Chemistry
More specific subject area	Biochemistry
Type of data	Tables
How data was acquired	Real time quantitative reverse transcription PCR
Data format	Analyzed
Experimental factors	The expression of genes involved in apoptosis was analyzed via qRT-PCR after treatment of cells with the cross-linking agents epichlorohydrin and diepoxybutane.
Experimental features	Cells were treated with cross-linkers, and reverse transcription and amplification were performed in a single reaction with a real-time PCR system. The comparative CTmethod was used to quantify gene expression in drug-treated versus untreated cells., with actin used as the endogenous control.
Data source location	N/A
Data accessibility	Data are within this article