Silencing CDK4 radiosensitizes breast cancer cells by promoting apoptosis
- Katie R Hagen†1,
- Xiangbin Zeng†1,
- Mi-Young Lee†1,
- Shannon Tucker Kahn†1,
- Mary Kathryn Harrison Pitner1,
- Sandra S Zaky1,
- Yuan Liu3,
- Ruth M O’Regan2,
- Xingming Deng1 and
- Harold I Saavedra1Email author
© Hagen et al.; licensee BioMed Central Ltd. 2013
Received: 3 April 2013
Accepted: 18 July 2013
Published: 25 July 2013
The discovery of molecular markers associated with various breast cancer subtypes has greatly improved the treatment and outcome of breast cancer patients. Unfortunately, breast cancer cells acquire resistance to various therapies. Mounting evidence suggests that resistance is rooted in the deregulation of the G1 phase regulatory machinery.
To address whether deregulation of the G1 phase regulatory machinery contributes to radiotherapy resistance, the MCF10A immortalized human mammary epithelial cell line, ER-PR-Her2+ and ER-PR-Her2- breast cancer cell lines were irradiated. Colony formation assays measured radioresistance, while immunocytochemistry, Western blots, and flow cytometry measured the cell cycle, DNA replication, mitosis, apoptosis, and DNA breaks.
Molecular markers common to all cell lines were overexpressed, including cyclin A1 and cyclin D1, which impinge on CDK2 and CDK4 activities, respectively. We addressed their potential role in radioresistance by generating cell lines stably expressing small hairpin RNAs (shRNA) against CDK2 and CDK4. None of the cell lines knocked down for CDK2 displayed radiosensitization. In contrast, all cell lines knocked down for CDK4 were significantly radiosensitized, and a CDK4/CDK6 inhibitor sensitized MDA-MB-468 to radiation induced apoptosis. Our data showed that silencing CDK4 significantly increases radiation induced cell apoptosis in cell lines without significantly altering cell cycle progression, or DNA repair after irradiation. Our results indicate lower levels of phospho-Bad at ser136 upon CDK4 silencing and ionizing radiation, which has been shown to signal apoptosis.
Based on our data we conclude that knockdown of CDK4 activity sensitizes breast cancer cells to radiation by activating apoptosis pathways.
KeywordsCDK4 CDK2 Radiation Bad Radioresistance Radiosensitization Breast cancer Apoptosis
Breast cancer is the most common malignancy and the major cause of cancer related deaths of women in industrialized countries . Fortunately, the discoveries of various biomarkers have greatly improved the treatment of breast cancer patients . For example, breast cancer patients that express progesterone receptor (PR) and estrogen receptor (ER) (luminal A breast cancers are ER+PR+HER2- or ER+PR+HER2+, while luminal B are ER+PR+HER2-) are treated with endocrine therapies and/or with the antibody Herceptin, in combination with chemotherapy and/or radiotherapy . Regrettably, the defining characteristic of triple negative breast cancers (a subtype of basal breast cancers) is the absence of ER, PR and HER2, rendering those cancers insensitive to some of the most effective therapies . ER-PR-HER2- and ER-PR-HER2+ patients show decreased survival relative to luminal subtypes [5, 6].
Radiotherapy is one of the cornerstones in the treatment of patients with breast cancer [7, 8]. Radiation greatly reduces the risk of recurrence in women with ductal carcinoma in situ [9, 10] and in breast cancer patients who are lymph node positive [11–13]. The outcomes following chemotherapy vary depending on the subtype: for example, ER-PR-HER2- and ER-PR-HER2+ breast cancers respond better than luminal subtypes to anthracycline-based chemotherapies . In contrast, all subtypes respond equally well to radiation . Regardless of treatment, ER-PR-HER2- or ER-PR-HER2+ breast cancers are more likely to recur upon breast conserving therapies or following mastectomy and radiation [16–19]. Even though radiotherapy shows promise in the treatment of all breast cancer subtypes, radiotherapy is associated with increased risk of cardiovascular disease . In addition, breast tumors can acquire radioresistance [21, 22]. Finding agents that sensitize malignant cells to radiation would increase tumor response while minimizing toxicity to surrounding organs by lowering effective therapeutic doses.
The G1/S phase regulatory machinery includes cyclins and cyclin kinase inhibitors (CKIs) that regulate the activities of the G1 phase CDKs [23, 24] and the Rb/E2F pathway [25, 26]. Besides being distinguished by their ER, PR, or Her2 status, the various breast cancer subtypes exhibit deregulated expression of proteins that ensure progression through the G1/S phase of the cell cycle. ER-PR-HERr2- breast cancers are more likely to overexpress mutant p53, E2F3, p16, and cyclin E and display lower levels of cyclin D1, Bcl2, and Rb relative to other breast cancer subtypes [5, 6, 27, 28]. Cyclin D1 overexpression is more often found in tumors with wild type p53, higher grades of differentiation, and expression of ER or PR [29–32]. Specifically, 53% PR+ and 58% ER+ breast cancer patients overexpress cyclin D1, while a smaller, but significant fraction of ER- and PR- breast cancers (21% and 31% respectively) overexpress cyclin D1 .
Several studies have shown that G1/S phase regulatory molecules may drive lower survival rates in patients and resistance to adjuvant therapies. Deregulated expression of cell cycle molecules that specifically modulate CDK2 kinase activity has been associated with poor prognosis of breast cancer patients. For example, high cyclin A expression in metastatic breast cancer correlates significantly with a shorter time to first relapse and is a prognostic factor in early stage ER+ breast tumors [34, 35]. Additionally, high cyclin E expression predicts a poor prognosis in breast cancer [36, 37]. Unregulated CDK2 activity may result in poorer survival due to the modulation of responses to various therapeutic agents. For example, low expression of p27Kip1 and/or cyclin E overexpression predicts early relapse in patients treated with adjuvant therapy that includes tamoxifen [38, 39], doxorubicin, cyclophosphamide , and Herceptin . Increased CDK2 kinase activity drives Herceptin resistance in vitro and in vivo [41, 42].
Deregulated CDK4/CDK6 activities have also been associated with decreased survival and resistance to various therapies. Cyclin D1 is overexpressed in over 50% of breast cancers [43, 44]. The oncogenic capacity of cyclin D1/CDK4/CDK6 in experimental models, including mouse models of mammary carcinogenesis has been established [45–47]. However, the role of cyclin D1/CDK4/CDK6 in breast cancers is highly controversial. For example, increased cyclin D1 mRNA and amplification of CCND1 in ER+ breast cancers strongly correlated with increased risk of relapse, local recurrence, metastasis, and death [30, 31, 48], and ER- patients with cyclin D1 overexpression display shorter overall survival . In contrast, other studies showed that patients overexpressing cyclin D1 (including ER+ tumors and unclassified breast tumors not falling within the canonical HER2, PR, ER classification) were less likely to recur following treatment and displayed longer survival [32, 49–51]. Similar to cyclins E and A, there is experimental evidence as to the involvement of cyclin D in resistance or sensitivity to various therapies. Patients within the ER+ subgroup who received endocrine therapy for their primary or recurrent breast cancers showed an association between high cyclin D1 and a shorter response duration [30, 52, 53]. In addition, in vitro studies showed that overexpressed cyclin D1 leads to resistance to antiestrogens . While some studies showed that ectopic cyclin D1 expression is directly involved in radioresistance and the poor prognosis of various carcinomas after radiotherapy [55–59], others found that ectopic expression of cyclin D1 markedly increases cell sensitivity to apoptosis induced by various agents including ionizing radiation [60–63]. In addition, chemical inhibition of CDK4 and CDK6 synergizes with Herceptin and tamoxifen treatments .
Although CDK4 and CDK2 are promising targets in cancer therapeutics, their role in the response of ER-PR-HER2+ or ER-PR-HER2- breast cancer cells to ionizing radiation is controversial and not extensively explored. We present data showing that knockdown of CDK4, but not of CDK2, imparts radiosensitivity to breast cancer cells and normal mammary epithelial cells by signaling an apoptotic program.
G1/S phase regulatory molecules are ectopically expressed in radioresistant breast cancer cells
IC 50 of Irradiation in non-transfected human breast cell lines
P value 1
Because the breast cancer cell lines overexpressed cyclin D1, and cyclin A protein levels were elevated following irradiation, we tested whether knockdowns of their respective kinase partners CDK4 and CDK2 might alter the relative radioresistance of various breast cancer cell lines. Therefore, we generated ER-PR-Her2- and MCF10A cell lines stably expressing shCDK2 or shCDK4. All cell lines showed consistently stable decreases in the protein level of CDK2 or CDK4 (Figure 1C). MCF10A and HCC1806 cells displayed complete downregulation of CDK4, while MDA-MB-468 and MDA-MB-231 cells showed a partial, but almost complete knockdown of CDK4. In addition, HCC1806 and MDA-MB-231 cells displayed complete knockdown of CDK2, while MCF10A and MDA-MB-468 cells displayed a partial, but significant knockdown of CDK2. Importantly, knockdown of CDK2 did not affect CDK4 levels and vice versa, demonstrating that specific knockdowns were achieved. To assess whether silencing of CDK4 altered radioresistance in other breast cancer subtypes, we generated HCC1954 and SKBR3 cells (both ER-PR-HER2+ cell lines) knocked down for CDK4 (Figure 1C). Similarly to ER-PR-HER2- and MCF10A cells, CDK2 levels in ER-PR-HER2+ cells were not affected by knockdown of CDK4.
Downregulated CDK4 sensitizes breast cancer cells to radiation
IC 50 of irradiation in breast cancer cell lines with CDK2 or CDK4 knockdown
P value 1
Radiosensitivity imparted by downregulated CDK4 is not caused by cell cycle blocks
Comparison of BrdU between control (pLKO.1) and treatment (shCDK4) groups
Comparison of phospho-Histone 3 between control (pLKO.1) and treatment (shCDK4) groups
Downregulation of CDK4 does not alter rates of DNA break repair
Comparison of γ-H2AX between control (pLKO.1) and treatment (shCDK4) groups
Irradiated breast cancer cell lines silenced for CDK4 display increased frequencies of apoptosis
To establish whether the increases in apoptosis caused by radiation in cells silenced for CDK4 could be detected with an independent apoptosis marker, cells were subjected to Western blots using cleaved PARP (Figure 4C). In accordance with cleaved caspase-3 immunocytochemisty, higher levels of cleaved PARP were detected in MCF10A (6 to 11 fold changes) cells silenced for CDK4 compared to vector control. Consistent with the small increases in apoptosis with cleaved caspase-3, cleaved PARP levels were slightly elevated in MDA-MB-231 silenced for CDK4 relative to pLKO.1 controls. Likewise, we detected slightly elevated cleaved PARP levels in MDA-MB-468 shCDK4 cells irradiated with 2 Gy relative to MDA-MB-468 pLKO.1 cells. In contrast, no difference was found in cleaved PARP between MDA-MB-468 shCDK4 and MDA-MB-468 pLKO.1 cells upon irradiation with 4 Gy.
To establish whether radiosensitization also occurred through autophagy, we probed Western blots with an antibody against LC3A/3B. In MCF10A cells, only the lower band, which is the indicator of autophagy, was detected with no great difference among samples. Both inactive and active bands were observed in MDA-MB-231 and MDA-MB-468 cells, again with no great differences among samples (Additional file 4A). Finally, to investigate if the CDK4/6 kinase inhibitor, PD0332991, has a similar effect on inducing apoptosis or autophagy, MCF10A, MDA-MB-231 and MDA-MB-468 cells were treated at their respective IC50s  of 100 nM, 500 nM, and 1000 nM PD0332991, irradiated after 24 hours, and analyzed after 48 hours for cleaved PARP (Figure 4D). Unlike biological knockdown, levels of cleaved PARP did not increase in MCF10A pLKO.1 cells treated with PD0332991. Instead, high levels of cleaved PARP (2 to 3 fold) were detected in MDA-MB-468 pLKO.1 cells treated with PD0332991 compared to untreated cells. Again, LC3A/3B levels were not changed greatly among any of the cell lines, and did not change in the presence or absence of CDK4 (Additional file 4B). These results demonstrate that knockdown of CDK4 and chemical inhibition of CDK4/CDK6 lead to different outcomes, as the inhibitor is protective of apoptosis in irradiated MCF10A and MDA-MB-231 cells, while promoting apoptosis in MDA-MB-468 cells. Overall, the results also demonstrate that the primary mechanism of cell death in this system is through apoptosis and not autophagy.
Knockdown of CDK4 results in reduced phosphorylation of BAD (ser136) and upregulation of protein phosphatase type 2A (PP2A)
Silencing the catalytic subunit of PP2A does not lead to increased PARP cleavage in irradiated cells silenced for CDK4
The G1 phase CDKs, (CDK2, CDK4, and CDK6) display specific functions in terms of development and redundant functions in terms of cell cycle regulation [23, 24, 83]. The redundant nature of the G1 phase CDKs is best illustrated by work showing that cells can proliferate in the absence of CDK2 and CDK4, and that CDK1 activity is required for cell cycle progression [84, 85]. However, injured hepatocytes do not require CDK1 to proliferate . Another important function of the CDKs is in DNA repair. Specifically, cyclin D1, CDK4 [58, 59, 87], cyclin A1, and CDK2 have been implicated in DNA repair [88–92]. In fact, the activities of all CDKs are required for efficient DNA repair . Even though CDK2 and CDK4 are largely dispensable in the entry and progression through S phase, they are important mediators of mammary tumorigenesis in mice, as ablation of either CDK2 or CDK4 can prevent mouse mammary tumorigenesis triggered by HER2/Neu [94, 95]. Also, CDK2 and CDK4 can differentially mediate centrosome amplification depending on the oncogenic or cellular context . While cyclin D1/CDK4 specifically mediates centrosome amplification triggered by H-RasG12D, H-RasG12D&c-Myc , and in HER2+ breast cancer cells , silencing of cyclin E or CDK2 have no impact on centrosome amplification in the HER2/Ras systems. In contrast, both CDK2 and CDK4 mediate centrosome amplification and chromosome instability in p53 deficient cells .
Overall, the ability of CDK2 and CDK4 to mediate various malignant phenotypes that play important roles in cancer biogenesis has prompted the development of CDK2 or CDK4 specific inhibitors. Those inhibitors have proven to be effective in a variety of breast cancer cell lines. For example, ER+ luminal cells that overexpress cyclin D1 and Rb and display downregulated p16 respond well to cell cycle inhibition by the CDK4/CDK6 specific inhibitor PD-0332991, while non-luminal/basal cells are refractive to growth inhibition . It has been reported that the Rb status dictates the early responses to that inhibitor . Even though the CDK4/CDK6 inhibitor has shown promise in preclinical trials, cells acquire resistance through the upregulation of CDK2 activity . A major limitation of PD-0332991 is that while it is effective in extinguishing mammary tumors, its ability to block the cell cycle interferes with chemotherapy [99, 100].
In the present study, we addressed whether the G1/S cell cycle regulatory machinery influences radioresistance in MCF10A, ER-PR-HER2-, and ER-PR-HER2+ cells. There is a precedent for the role of ectopic CDK2 and CDK4 activities in imparting resistance or sensitivity to radiotherapy. For example, knockdown of cyclin E synergizes with doxorubicin to enhance radioresistance in breast cancer cells lines . Induction of cyclin A has previously been observed in cells treated with UV or irradiation [91, 102]. Cyclin A null cells are radiosensitive and display impaired double strand break repair; cyclin A/CDK2 is involved in DNA repair following irradiation by phosphorylating KU-70 .
Various mechanisms have been postulated to explain how cyclin D1/CDK4 leads to radioresistance or radiosensitivity of various cells. Inducible expression of cyclin D1 in MCF7 breast cancer cells leads to radiosensitivity through activation of the p53 pathway . Low dose irradiation promotes the free, cytoplasmic cyclin D1 accumulation in human keratinocytes, correlating with radioresistance; in this context, low level radiation disrupted the interaction of cyclin D1 with 14-3-3ζ . Cytoplasmic cyclin D1 then interacts with Bax, suppressing the ability of Bax to induce apoptosis. In another example, long term fractionated irradiation of the human cancer cell lines HepG2 and HeLa induced radioresistance . This radioresistance correlated with upregulation of cyclin D1 due to the stabilization of cyclin D1 by prevention of its proteolysis, achieved by the DNA PK/AKT/GSK3β pathway. In addition, they first proposed that ectopic expression of cyclin D1 leads to increased radioresistance in breast cancer cells by enhancing DNA repair in the radioresistant cells, as downregulation of cyclin D1 in this system abrogated enhanced DNA repair, resulting in radiosensitivity. This group showed using chemical inhibitors that cyclin D1 dependent radioresistance is reversed by AKT or CDK4 chemical inhibitors. A recent report also demonstrated that enhanced cyclin D1 results in enhanced DNA repair and radioresistance . This report postulates that cyclin D1 promotes DNA repair by interacting with RAD51 within DNA repair foci; this interaction is promoted by radiation.
Our studies demonstrate that knockdown of CDK4 acts as a potent radiosensitizer, independently of the breast cancer molecular subtype. In our studies, we tested whether radiosensitization was a consequence of cell cycle inhibition by silenced CDK4, altered DNA break repair, or through the activation of an apoptotic program. We used various assays to show that knockdown of CDK4 did not influence the cell cycle of non-irradiated or irradiated cells. This is consistent with our previous observations that in MCF10A cells expressing control pLKO.1, H-RasG12V, H-RasG12V&c-Myc, or in HER2+ breast cancer cells, knockdown of CDK4 does not greatly influence DNA replication or cell cycle progression [76, 97]. This is also consistent with our data showing that ablation of CDK4 does not alter the cell cycle of p53−/− mouse embryonic fibroblasts . In contrast, co-inhibition of CDK4 and CDK6 using a chemical inhibitor interferes with the cell cycle, leading to protection against chemotherapy [99, 100]. Our studies conducted with the PD0332991 CDK4/CDK6 inhibitor revealed a similar story, as treatment of MCF10A or MDA-MB-231 protected against radiation-induced apoptosis. This is contrary to our studies revealing that knockdown of CDK4 synergizes with radiation to induce apoptosis in breast cancer cells and MCF10A controls. The only cell in which radiation synergized with inhibition of CDK4/CDK6 was MDA-MB-468, a cell that is insensitive to growth inhibition by PD0332991. However, we do not think that insensitivity to growth inhibition is the sole cause of the sensitivity to apoptosis triggered by PD0332991 treatment, as we treated all cell lines exactly at IC50 to prevent complete growth inhibition. One potential explanation for the differences between knockdown of CDK4 and inhibition with PD0332991 is that while the chemical inhibits CDK4/CDK6 activity, the shCDK4 may disrupt the protein:protein interactions of CDK4 with cyclin D, p21, p27, p15, p16, p18 and p19, leading to modulation of the activities of other CDKs.
In contrast to studies showing that increased cyclin D1 activity results in radioresistance through increasing cellular DNA repair capacity, all breast cancer cells tested in this study efficiently repaired DNA breaks irrespective of CDK4 levels, as indicated by the efficient clearance of H2AX foci after radiation. A recent study is consistent with our results, as chemical inhibition of CDK4 and CDK6 in breast cancer cells did not influence double-strand break repair after irradiation . We speculate that the differences between cyclin D1 and CDK4/CDK6 inhibition in relation to DNA repair is because cyclin D1 displays functions that are independent of CDK4. For example, it can bind the promoters of genes involved in chromosome instability .
The presence of increased levels of cleaved caspase-3 and of cleaved PARP in all irradiated cells down regulated for CDK4 indicated that apoptosis was a significant driver of radioresistance in mammary epithelial and breast cancer cells. Although apoptosis is high in unirradiated MCF10A cells silenced for CDK4, levels of apoptosis rise significantly in those and in the breast cancer cells silenced for CDK4 upon irradiation. This suggests synergy between downregulation of CDK4 and irradiation in regards to apoptosis. To establish how absence of CDK4 cooperates with radiation to enhance cell death, we screened various Bcl family members. No changes in the levels of phosphorylation of the antiapoptotic members Bcl2 or Mcl1 were observed in the cells. In contrast, levels of phosphorylated Bad ser136 were sharply diminished Even though dephosphorylation of Bad in serine 136 is associated with apoptosis, we observe increased phosphorylation in irradiated shCDK4 MCF10A cells. The regulation of apoptosis is complex, involving various protein/protein interactions, as well as the post-translational modification of numerous proteins. Further studies must be performed in order to establish why MCF10A cells silenced for CDK4 display phosphorylated Bad Ser 136, and still apoptose. Bad ser136 phosphorylation is reversed by various phosphatases, including PP2A and PP1α [81, 82]. In fact, silencing of CDK4 resulted in higher levels of PP2A, suggesting that it specifically dephosphorylates Bad ser136 upon irradiation PP2A is a protein complex with many cellular functions, including the regulation of apoptotic and mitogenic pathways [104–106], as well as the modulation of DNA repair . Inhibition of PP2A by DNA tumor viruses is tightly linked to cellular transformation . Also, the dephosphorylation of Bcl2 by PP2A promotes its anti-apoptotic activity; this event may enhance Bcl2’s oncogenic potential . In wide contrast, PP2A’s proapoptotic activities have been mapped to its ability to dephosphorylate Bad [81, 82] and Bax . We did not observe significant dephosphorylation of Bcl2 in any of the cell lines, suggesting that upregulation of PP2A in breast cancer cells exclusively dephosphorylates Bad ser136 without significantly affecting other Bcl family members. Thus, our observation that downregulation of CDK4 resulted in decreased phosphorylation of Bad ser136 suggests that this event is primarily responsible for apoptosis and radioresistance. Additional experiments are required to address how CDK4 regulates PP2A levels; that regulation may result from increased protein stability, increased degradation, or enhanced de-repression of E2F target genes upon CDK4 silencing. Another important future area of exploration is whether and how the apoptotic program triggered by downregulation of CDK4 in conjunction with radiation is rooted in Rb/E2F dependent transcription.
Unfortunately, we were unable to show that ectopic expression of PP2Ac radiosensitizes breast cancer cells, or that its inhibition modulates radiation induced apoptosis in breast cancer cells. We conclude that apoptosis in cells knocked down for CDK4 is independent of PP2A. Further experiments are required to establish pathways signaling apoptosis in irradiated breast cancer cells knocked down for CDK4.
We conclude that stable knockdown of CDK4 enhances the effects of radiation by enhancing apoptotic pathways. This result has implications for future therapies, as patients can be treated with radiation or chemotherapeutic agents and with doses of CDK4/CDK6 inhibitors that do not block the cell cycle. In that scenario, suppression of CDK4/CDK6 activity would allow cell cycle progression, while synergizing with radiation to increase cell death.
Materials and methods
A human mammary epithelial cell line (MCF10A) and breast cancer cell lines (ER-PR-HER2-:HCC1806, HS578, MDA-MB-231, MDA-MB-468; ER-PR-HER2+: SKBR3 and HCC1954), were purchased from ATCC (Rockville, MD), or obtained through collaborators. The molecular markers and various other characteristics of the breast cancer cells used in this study have been previously described . HCC1806, HCC1954, and SKBR3 cells were cultured in RPMI supplemented with 10% fetal bovine serum (FBS). HS578, MDA-MB-231 and MDA-MB-468 cells were cultured in DMEM supplemented with 10% FBS. MCF10A cells were cultured in DMEM/F12 supplemented 10% FBS, 20 ng/ml EGF, 100 ng/ml cholera toxin, 0.01 mg/ml insulin, and 500 ng/ml hydrocortisone. All cell lines were cultured at 37°C in a humidified atmosphere (5% CO2).
Colony forming assay
Two thousand five hundred cells were plated per well in a 6-well culture plate or one thousand cells were plated per well in a 12-well cell culture plate (Day 0) in triplicates. After 24 hours (Day 1), cells were irradiated at a dose of 0, 1, 2, 4, 6, or 8Gy in an X-RAD 320 irradiator (Precision X-Ray, CA). After an average of 8 days, cells were fixed with 75% ethanol and stained with 1% crystal violet, and colonies containing more than 50 cells (established by microscopy) were counted manually. Plating efficiency was calculated by dividing the number of colonies in the unirradiated group divided by the number of colonies plated and multiplying by 100.
Lentiviral shRNA transduction
The shRNA constructs against CDK2 or CDK4 were cloned into the lentiviral vector pLKO.1, which are commercially available from OPEN Biosystems. Lentiviral infections were done as described in our publications . Briefly, cells were plated in regular medium at 50% confluence on day 1. This was followed by changing into medium containing 5 μg/ml polybrene and infection of cells by adding undiluted shRNA lentiviral particles to the culture on day 2. Stable clones expressing the shRNA were obtained by splitting cells 1:5 and culturing in medium containing puromycin.
Immunofluorescence was performed by following our published protocols [73, 97]. Cells were plated at 4×104 per well into two-well tissue culture chamber slides and received 2 Gy irradiation after cells attached. Cells were then fixed in cold 4% paraformaldehyde at different times postirradiation (from 0 to 48 hr), washed in PBS, permeabilized in a 1% NP-40–PBS solution, and blocked in 10% goat serum. Commitment to apoptosis was detected by using an antibody against cleaved caspase-3 (9661, Cell Signaling). DNA breaks were detected using an antibody against phosphorylated histone 2A variant X (γ-H2AX) (ab2893, Abcam). Cell proliferation was examined using an antibody against Ki-67 (ab15580, Abcam). The BrdU assay was used to detect cells in S phase and was done by pulsing cells with 20 μM BrdU (51-7581KZ; BD Pharmingen) 30 minutes before harvest and detecting with an anti-BrdU antibody (NA61; Calbiochem). To detect mitosis, cells were plated at 1×104 per well into four-well tissue culture chamber slides and received 0, 2 and 4 Gy irradiation 24 hours after plating and incubated 48 hours and phosphor-histone 3 (Ser 28, 9713, Cell Signaling) antibody was used to detect mitosis. Secondary antibodies are conjugated with Alexa Fluor 555 or Alexa Fluor 488. Cells were counterstained with DAPI. Frequencies of the various phenotypes are done by counting the number of antigen-positive cells divided by the total number of cells; unless otherwise indicated, we count at least 200 cells per group. For mitosis, over 300 cells per group were counted.
Western blotting was performed according to published protocols [73, 97]. Protein lysates were obtained by incubating cells in lysis buffer (50 mM HEPES, 250 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% NP-40, 10% glycerol) containing protease inhibitors (04693124001, Roche) for 30 min at 4°C. Calyculin phosphatase inhibitor (Millipore 19-139v) was added to the culture medium at a concentration of 100 nM, 30 minutes before harvest for the detection of phosphorylated proteins. Samples were denatured at 95°C for 5 min in sodium dodecyl sulfate (SDS) sample buffer, resolved by SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad). The blots were incubated in blocking buffer (5% nonfat dry milk in 1X Tris-buffered saline plus 0.1% Tween 20 [TBS-T]) for 1hr and were then probed overnight at 4°C with the primary antibodies. The blots were then rinsed in 1X TBS-T and incubated with the appropriate horseradish peroxidase conjugated secondary antibodies at room temperature. The blots were then rinsed in 1X TBS-T and the antibody-antigen complex was visualized using Lumigen TMA-6 (RPN2135v1 and RPN2135v2; GE Healthcare).
Antibodies and inhibitors
The antibodies used in the various Western blotting experiments were as follows: CDK2 (sc-163, Santa Cruz), CDK4 (2906, Cell Signaling), p21Waf1 (sc-397, Santa Cruz), p27Kip1 (sc-528, Santa Cruz), β-actin (4970, Cell Signaling), cyclin A (ab38, Abcam), cyclin D1 (2922, Cell Signaling), cyclin E (sc-481, Santa Cruz), Phospho-Bcl-2 ser70 (2827, Cell Signaling), Bcl-2 (OP60, Calbiochem), Phospho-Mcl ser159/thr163 (4579, Cell Signaling), Mcl-1 (sc-819, Santa Cruz), Phospho-Bad ser136 (06–846, Upstate), Bad (1541–1, Epitomic), Phospho-AKT Ser 473 (Cell Signaling), PP1α (2582, Cell Signaling), PP2A (2259, Cell Signaling) and PP2B (2614, Cell Signaling), cleaved PARP (Asp214, 5625, Cell Signaling). The CDK4/6 inhibitor PD0332991 (Axon1505) was purchased from Axon Medchem. When indicated, ImageJ software was used to quantify protein levels of Western blots (NIH).
Cells were irradiated at 2 Gy, pulsed with BrdU (added at a final concentration of 10 μM 30 min prior to harvest) and harvested at various time points (6, 12, and 24 hours). Timepoints were chosen based on our preliminary and supplementary data. A unirradiated control was also collected. 1×106 cells were transferred to 5ml round bottom tubes (352235, BD), and cell staining was performed according to the protocol included with the “Apoptosis, DNA damage and Cell Proliferation kit” (562253, BD) or stained with 7-AAD (559925, BD Biosciences). BrdU was detected using PerCP-Cy™5.5 Mouse Anti-BrdU (51–9007682, BD), PARP cleavage was identified with PE Mouse Anti-Cleaved PARP (Asp214) Antibody (51–9007684, BD) and γH2AX was distinguished using Alexa Fluor® 647 Mouse Anti-H2AX (pS139) (51–9007683, BD). A LSRII flow cytometer (BD) was used to read each sample. Compensation was performed using Anti-Mouse Ig, κ/Negative Control (FBS) Compensation Particles Set (BD, 552843) and samples were analyzed using Flowjo v7.6.5 and v10.4 (Tree Star).
For the colony forming assay, a dose response analysis was conducted, in which the dose response relationship was assumed to be the commonly used four parameter logistic function . The data was normalized relative to the baseline dose level=0 Gy (no radiation), and the percentage of control was used as the primary outcome in the analysis. IC50, the radiation dose that is required for 50% reduction from baseline dose level, was the parameter of interest and compared accordingly. The higher value of IC50, the higher the resistance. A proper data transformation, Box- Cox transformation,  was applied to assure the fitted model meeting the statistical assumptions. All data management and statistical analyses were done in R . T-tests were done in Excel. The statistical significance level of all tests was set at 0.05.
For the flow cytometry assays (DNA content and BrdU) the data was aligned by the six-hour gates. The two measurements (percentages of positive cells) per each run are averaged at each time point and the averaged measurement at each of 6 hours, 12 hours and 24 hours after radiation is divided by the averaged measurement at baseline. The mean percentage of the positive cells relative to no radiation is used for analyses, named fold-change to no radiation. The main goal of this data analysis is to compare the fold change between control and treatment groups at each time point, within each cell line, biomarker, and gate. The fold change is compared between control group and treatment group (pLKO.1 vs. shCDK4) at each time point within each cell line, biomarker, and gate using ANOVA (Analysis of Variance) to estimate the mean and standard error for the fold change to non-radiation. For the cleaved caspase-3 assay, observations (number of positive or negative cells) are combined within each radiation dose of 2 Gy, 4 Gy or no radiation treated cells for each control group and treatment group and then the Chi-square test or Fisher’s exact test is used to compare the proportions of positive and negative cells between treatment and control groups. Analysis unit is a cell. The significance levels are set at 0.05 for all tests. The SAS statistical package V9.3 (SAS Institute, Inc., Cary, North Carolina) is used for data managements and analyses.
No research on humans or animals were conducted. All human cell lines were purchased from the publically available American Type Culture Collection (ATCC), or obtained through collaborators.
We would like to thank Dr. Rita Nahta for various Her2+ breast cancer cell lines, and Dr. Jing Chen for 293T cells and helper plasmids. We thank Dr. Sungjin Kim from the Biostatistics and Bioinformatics Shared Resource at Winship Cancer Institute for statistical analysis. This research project was supported by R01 CA151521 from the National Institutes of Health, the Georgia Cancer Coalition, The Sindab Triple Negative Breast Cancer Foundation, and by the Emory University Integrated Cellular Imaging Microscopy Core of the Winship Cancer Institute comprehensive cancer center grant, P30CA138292.
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