Microarray determination of Bcl-2 family protein inhibition sensitivity in breast cancer cells

Cell lines and reagents

The four breast cancer cell lines examined were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Each cell line was resuscitated for less than three months in our laboratory. The BT-20 line was maintained in Dulbecco's Modified Eagle's medium (DMEM) along with Ham's F-12 Nutrient Mixture in a 1:1 ratio (Hyclone Laboratories, Logan, UT, USA) with 10% fetal bovine serum (FBS), 2% penicillin/streptomycin, 10 mmol/L HEPES, 1% sodium pyruvate, 2% sodium bicarbonate and 1% non-essential amino acids. T47-D, MDA-MB-468 (MDA-468) and MDA-MB-231 (MDA-231) were maintained in DMEM, which contained 10% FBS, 2% penicillin/streptomycin, 10 mmol/L HEPES, 1% sodium pyruvate, 2% sodium bicarbonate and 20 mmol/L glucose. The T47-D line was derived from infiltrating ductal carcinoma whereas the other cell lines were of adenocarcinoma origin.

Drug treatment and irradiation

The small molecule pan Bcl-2 inhibitor, obatoclax mesylate (GX15-070), was purchased from Selleck Chemicals LLC (Houston, TX, USA). Obatoclax was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 1 mmol/L and working dilutions were prepared in DMEM medium. Untreated control cells had the equivalent amount of DMSO added as the agent was diluted. Mock-irradiated cells (0 Gy) in plates were manipulated in the same way as those that received 2 Gy dose of x-irradiation. The X-ray source was an RS 2000 X-ray Biological Irradiator (Rad Source Technologies, Inc., Suwanee, GA, USA). Four hours after irradiation, cells were treated with 1 μmol/L of obatoclax in a 37°C 5% CO₂ incubator. Assays were performed at 48 h postirradiation.

Microarrays

SABiosciences apoptosis arrays were used as described by the manufacturer (SABiosciences, Qiagen Inc., Valencia, CA, USA). The plates were read on a Roche LightCycler 480 System. The values for the four samples on each plate were separated using the online software at the SABioscience website.

To control for minor DNA concentration differences between samples, all values were normalized in comparison to the average of the five housekeeping genes on the micro-array. All samples were compared with normal breast HMECs and normalized by the difference between the housekeeping gene averages relative of the HMEC controls.

Further normalization controls: If the average of the housekeeping genes differed by more than 2 (log2), that entire sample was discarded. Additionally, if any individual housekeeping gene was missing or off scale, that individual housekeeping gene was excluded, except where we had multiple samples from the same DNA sample. In that case the missing housekeeping value was replaced by the average of the others from the same DNA and same housekeeping gene. An average of three replicates was used for each cell line except for MDA-468 where there were four replicates.

Western blotting

The level of protein expression was determined by immuno-blotting. Proteins were extracted from cell extracts by lysis in RIPA buffer and a Complete Mini Protease Inhibitor tablet (Roche Diagnostics, Indianapolis, IN, USA). The 7.4 pH RIPA buffer was comprised of 150 mmol/L sodium chloride, 1.7 mmol/L monobasic sodium phosphate, 9.1 mmol/L dibasic sodium phosphate, 0.5% sodium de-oxycholate (Fisher Scientific Chemicals, Fair Lawn, NJ, USA), 0.1% sodium dodecyl sulfate and 1% Nonidet P-40 (Sigma Chemical Co., St. Louis, MO, USA). Proteins were separated on 4–12% polyacrylamide gels and transferred to polyvinylidene difluoride membranes, blocked with 5% milk block overnight, rinsed with TBS/Tween-20 and hybridized with antibodies specific to human: Bcl-2, Bcl-xL, Mcl-1, A1 and Bcl-w (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at a 1:200 dilution for 1.5 h at room temperature. SeeBlue Plus2 Prestained Standard (Life Technologies, Grand Island, NY USA). The loading control was β-actin, clone AC15 (MA1-91399, Thermo Scientific Inc., Rockford, IL, USA).

Antibodies were detected on the membranes with an ECF Western Blotting Kit (Amersham Biosciences, Piscataway, NJ, USA) and scanned on a Storm 840 Phosphorimager (Molecular Dynamics, Sunnyvale, CA, USA). Each experiment was performed at least twice and results were determined using ImageJ64 software (rsbweb.nih.gov/ij/ download.html). All Western blot images were quantified using unsaturated densitometry scans. Western blot band densities were quantified with ImageJ64 software and normalized by re-probing each membrane with an antibody to β-actin as a loading control.

Statistical analysis

Error bars on graphs represent standard errors of the mean. The t-test and the Newman–Keuls Multiple Comparison Test were performed using GraphPad (Prism) graphing software.

Flow cytometric and fluorescent microscopic analysis of apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling

Treated and control cells were assayed by terminal deoxy-nucleotidyl transferase dUTP nick end labeling (TUNEL) staining for late apoptosis.

All steps of the TUNEL procedure were performed at room temperature unless otherwise specified. Cell lines MDA-231 and BT-20 were seeded in a six-well plate at a concentration of 3 × 10⁵ cells per well in 1 mL of corresponding media and the T47-D and MDA-468 cells were plated at 2 × 10⁵ cells per well. The six-well plates were placed in a 37°C 5% CO₂ incubator for 24 h. After 24 h, the cells were irradiated at a 2 Gy dose of radiation with the RS 2000 X-ray Biological Irradiator. The plates were set up to have both irradiated samples (2 Gy) and mock irradiated samples (0 Gy) handled in the same manner. Four hours after irradiation, cells were treated with 1 μmol/L obatoclax for an additional 44 h in a 37°C 5% CO₂ incubator. Cells were collected 48 h postirradiation. Acquisition was with a BD LSRII flow cytometer with a 488 nm argon ion source and FACSDiVa software (Becton Dickinson Labware, Franklin Lakes, NJ, USA) and analysis with FlowJo software. Each apoptosis value was an average of two replicas for BT-20, three for MDA-468 and T47-D, and four for MDA-468. Immediately following flow cytometry the cells remaining in the tubes were cytospun onto glass slides and DAPI-containing vectashield (Vector Laboratories, Burlingame, CA, USA) was placed on the cytospun cells, coverslipped and visualized with an Olympus BX 60 fluorescence microscope. Four to five fields per slide with at least 100 cells total per slide were photographed at ×400 magnification and examined using MagnaFire software. The cells shown in Figure 5 are all from single fields, except for BT-20, for which a composite was made due to the low number of cells. Simple color balance and contrast adjustments were made using Adobe Photoshop and applied identically to all.

OPEN IN VIEWER

Figure 1

Western analysis of Bcl-2 family member proteins A1, Bcl-2, Mcl-1, Bcl-w and Bcl-xL. Total protein extracts from four different breast cancer cell lines were analyzed by Western blotting, with antibodies to each of five antiapoptotic Bcl-2 family proteins. β-Actin was used to control for loading differences (n = 2). Molecular weight standards are in lane s

OPEN IN VIEWER

Figure 2

Flow cytometry measurements of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stained cells. Representative TUNEL flow cytometry apoptosis assay: cell lines T47-D, BT-20, MDA-MB-231 and MDA-MB-468 were treated with dimethyl sulfoxide (DMSO) control, obatoclax, RT or RT plus obatoclax. (DMSO was added as control to all as in Figures 1 and 2.) The cells were stained with labeled TUNEL antibody and analyzed by flow cytometry 48 h later. Gating was performed as shown to remove small debris from the analysis

OPEN IN VIEWER

Figure 3

Quantification of apoptosis induced by either obatoclax or radiation therapy alone or in combination with obatoclax. (a) The values from three repetitions of data as in Figure 1a were averaged for each cell line. The differences between columns labeled a, b or c are statistically significant (P < 0.001) by the Newman–Keuls multiple comparison test. The error bars show the standard errors. Note that the amount of apoptosis induced by RT alone is approximately the same as the amount in the dimethyl sulfoxide controls. (b) The observed RT-plus-obatoclax apoptosis is significantly increased (P < 0.05) relative to what would be expected by simply adding the amount of apoptosis induced by obatoclax alone to the very small amount induced by RT alone. The apoptosis in the combined treatment is separated into the component induced by obatoclax alone (lower section) and the component induced by RT (A color version of this figure is available in the online journal)

OPEN IN VIEWER

Figure 4

The apoptosis resulting from the combined treatment is also quantified as the percentage of apoptosis induced by RT in the non-apoptotic cells remaining after obatoclax treatment (e.g. in MDA-231, it would be the percentage of the 50% non -apoptotic cells remaining after the obatoclax-only treatment (Figure 3a) that subsequently became apoptotic after RT). Note: in the percent remaining data, one of the three repetitions of the MDA-468 data was omitted from the average because well over 90% of the cells were apoptotic following obatoclax alone treatment in that repetition, and the addition of radiation did not increase apoptosis further. Statistics: a and b are significantly different by the Student's t-test (P < 0.05). Error bars are the standard errors (A color version of this figure is available in the online journal)

OPEN IN VIEWER

Figure 5

Fluorescent microscopy of terminal deoxynucleotidyl transferase dUTP nick end labeling stained cells prepared similarly to the ones used for flow cytometry in Figure 3

Microarrays

Reverse transcription polymerase chain reaction (RT-PCR) microarrays have been used to quantify mRNA from clinical samples. To determine whether RT-PCR micro-arrays could be used to determine relative sensitivity to obatoclax-induced apoptosis, we used the SABiosciences RT-PCR apoptosis microarray kit. This array contained primers to detect all the Bcl-2 family members, both pro-and antiapoptotic, as well as most of the XIAP family of genes that act downstream of the Bcl-2 family. The reader for the RT-PCR quantified doubling cycles (Ct) to reach a maximum rate of increase for each primer pair. We compared the normalized Cts (measured as log₂) for each of the apoptotic genes from the cell lines with similar normalized values for breast HMECs as a point of comparison.

The results are shown in Table 1. Highlighted in bold are the values where the average is at least one standard deviation above a single doubling of the expression level relative to the normal HMECs. The total as well as the equivalent family fold-overexpression increase is also shown. The total Bcl-2 family member's fold-increase was determined by adding the increases due to the individual Bcl-2 family member overexpressed mRNAs to give a number equivalent to the fold-increase that would be measured if a single gene were being overexpressed. Values less than a single doubling were ignored because these numbers mostly reflect noise.

OPEN IN VIEWER

Table 1

RT-PCR microarray quantification of Bcl-2 family member mRNA overexpression levels

	MDA-468 Ct (log2)	SD	MDA-231 Ct (log2)	SD	T47-D Ct (log2)	SD	BT-20 Ct (log2)	SD
Bcl-2	3.62	0.3	2.6	0.6	2.4	0.9	−0.8	0.3
A1	3.25	0.5	0.3	0.9	1.2	0.9	0.6	2.9
Bcl-xL	0.22	0.6	3.3	0.9	0.4	0.3	−2.1	2.7
Bcl-w	1.19	0.4	0.1	0.5	−0.6	2.3	−0.6	0.4
Mcl-1	0.6	0.5	−0.4	0.1	0.5	0.7	−0.8	0.8
Family total	6.90	0.7	5.9	1.0	2.4	0.9	0.0	0.0
Fold increase	21.9		17.4		5.2		0.0
Bak	0.76	0.4	0.9	0.8	0.0	2.9	−1.9	1.1
Bax	2.72	0.6	3.0	1.1	0.9	1.8	−0.2	0.8
Bim	3.99	0.5	2.0	1.1	0.6	1.4	2.7	0.9
Family total	6.70	0.7	5.0	2.2	0.0	0.0	2.7	0.9
Fold increase	22.5		12		0		0
NAIP	−2.85	0.8	2.9	0.3	1.5	0.8	0.9	0.4
BIRC2	2.22	0.6	1.2	0.2	0.8	0.6	−0.7	0.7
BIRC3	4.47	1.9	3.7	0.7	−0.2	1.5	1.4	1.1
XIAP	2.12	0.3	0.7	0.1	−0.3	1.3	1.5	1.0
BIRC6	2.36	0.2	0.6	0.8	0.6	0.4	1.7	0.9
Family total	8.10	1.0	6.6	0.8	0.0	0.0	0.0	0.0
Fold increase	29.1		20.1		0		0

Values, one standard deviation above one doubling of mRNA expression, are in bold. Family totals are the sum of the doublings for the bold values. Fold increase for each family is calculated as the sum of the individual fold increases, equivalent to a single gene being overexpressed. Multiple replicas (at least 3) of RT-PCR amplifications of the apoptotic gene expression levels from the four cell lines were quantified and compared with similarly determined normal mRNA levels from breast HMECs. RT-PCR, reverse transcription polymerase chain reaction

Table 1 shows that both MDA-468 and MDA-231 each overexpressed two of the five antiapoptotic Bcl-2 family members, but not the same two. Both overexpressed Bcl-2 itself. Additionally, MDA-468 also overexpressed A1 whereas MDA-231 overexpressed Bcl-xL. T47-D overex-pressed only Bcl-2 and to a lesser extent than MDA-468 (although at a similar level as MDA-231). BT-20 did not overexpress any of the antiapoptotic Bcl-2 family proteins.

In general, Bax and Bim, the proapoptotic Bcl-2 family members, were overexpressed to the same levels as the antiapoptotic Bcl-2 family members. Bax and Bim were significantly overexpressed by MDA-468 and MDA-231, whereas BT-20 overexpressed only Bim. T47-D overexpressed no single proapoptotic Bcl-2 family member significantly, but the sum total was significantly overexpressed. The antiapoptotic XIAP family members were also overexpressed in cell lines MDA-231 and MDA-468 in proportion to the over-expressed antiapoptotic Bcl-2 family members.

Western blots

The RT-PCR microarrays quantified mRNA levels, but not protein concentrations. To confirm that the overexpressed mRNAs resulted in elevated levels of their protein products, we used Western blots (Figure 1) to quantify the relative amount of the five commonly overexpressed antiapoptotic Bcl-2 family members: A1, Bcl-2, Bcl-xL, Mcl-1 (both bands), and Bcl-w in each of the cell lines. Labeled antibodies were used to quantify the amounts of the antiapoptotic Bcl-2 family members on the Western blots, which were then normalized by determining the amount of β-actin present in each sample. We compared the protein concentrations to BT-20 since it had the lowest level of mRNA expression.

The results of the Western analysis of protein concentrations (Table 2) were very similar to the microarray data for the mRNA expression levels. Breast cancer cell line MDA-468 had an elevated level of the proteins A1 and Bcl-2, and cell line MDA-231 had an elevated level of Bcl-2 and Bcl-xL. T47-D had high levels of Bcl-2 but relatively low levels of the other proteins, and BT-20 had the lowest levels of all the five proteins. The major difference between the microarray data and the Western blot data was that levels of Bcl-xL were higher in MDA-468, MDA-231 and T47-D compared with BT-20. Note that from the Western blots, we cannot rule out that some Bcl-2 family members were present at elevated levels (e.g. Mcl-1 in MDA-468).

OPEN IN VIEWER

Table 2

Bcl-2 family protein levels quantified by Western blotting

Westerns	MDA-468	SE	MDA-231	SE	T47-D	SE	BT-20
Bcl-2	1.53	0.38	2.03	0.40	1.35	0.31	1.00
A1	1.44	0.11	1.40	0.71	1.25	0.47	1.00
Bcl-xL	1.32	0.12	2.00	0.16	1.85	0.12	1.00
Bcl-w	1.31	0.31	1.1	0.04	1.11	0.13	1.00
Mcl-1	0.82	0.82	1.27	0.51	1.27	0.51	1.00

Values, one standard error above BT-20, are in bold. The results of two separate Western analyses for each Bcl-2 family member and each cell line were averaged and the standard error of the mean calculated. Note: In this table, the amounts are presented relative to BT-20, which had the lowest level in the Western blots, and also did not overexpress in the microarray analysis of the mRNAs

Apoptosis

To quantitatively determine the induction of apoptotic cell death by treatments with obatoclax and/or RT, we treated each cell line with obatoclax and/or RT, and quantified the apoptosis response by flow cytometric measurements of TUNEL-stained cells (Figures 2 and 3a). Obatoclax induced apoptosis in all four cell lines, but much more so in MDA-231 and MDA-468 than in BT-20 or T47-D cells (Figure 3a). In contrast (and as expected), RT alone (Figure 3a) induced almost no apoptosis in any of the cell lines, indicating that all four cell lines were resistant to RT-induced apoptosis. Figure 3b also showed the amount of apoptosis attributable to RT in the combined obatoclax-RT treatment as compared with what would be expected by simply adding the amounts of apoptosis produced by either treatment alone. When the obatoclax-treated cells were also treated with RT (Figure 3b), the level of apoptosis attributable to RT increased in all cell lines more than would be expected by a simple additive effect. Thus obatoclax sensitized cells to RT-induced apoptosis.

The values in Figure 3b, however, underestimated the interaction between obatoclax and RT. After the obatoclax treatment, a much lower percentage of MDA-231 and MDA-468 cells survived, compared with T47-D and BT-20. When we determined the percentage of cells induced to undergo apoptosis by RT in the population remaining after obatoclax treatment (Figure 4), a much greater percentage of the surviving MDA-231 and MDA-468 were apoptotic compared with the percentage of surviving BT-20 or T47-D cells. This was consistent with the results from the obatoclax alone treatment.

To qualitatively confirm the flow cytometry measurements, we also prepared TUNEL-stained cells for visualization by fluorescent microscopy (Figure 5). The microscopic examination analyzed fewer total cells, but without gating out dead cells or other anomalies. Additionally, microscopic examination permitted the analysis of the untreated controls, which in the flow cytometry data were considered to be near zero. In general, the microscopic observations were consistent with the flow cytometry data. Two of the cell lines, BT-20 and MDA-468, showed higher levels of apoptosis in the untreated controls than were expected from the flow cytometry data.