It has been suggested that p16 has a role in glucocorticoid (GC)-related apoptosis in leukemic cells, but the exact mechanisms have yet to be clarified. We evaluated the relationship between the GC response and p16 expression in a lymphoma cell line.
We used p16 siRNA transfection to construct p16-inactivated cells by using the B-cell lymphoblast cell line NC-37. We compared glucocorticoid receptor (GR) expression, apoptosis, and cell viability between control (p16+ NC-37) and p16 siRNA-transfected (p16- NC-37) cells after a single dose of dexamethasone (DX).
In both groups, there was a significant increase in cytoplasmic GR expression, which tended to be higher for p16+ NC-37 cells than for p16- NC37 cells at all times, and the difference at 18 h was significant (
These results suggest a relationship between GR expression and cell cycle inhibition, in which the absence of p16 leads to reduced cell sensitivity to DX.
Glucocorticoid (GC) is an important chemotherapeutic agent used to treat leukemias and lymphomas
Progression from the G1 to S phase of the cell cycle is regulated by a series of structurally related enzymes: cyclin regulates the activation of cyclin-dependent kinases (CDKs); CDKs regulate the retinoblastoma protein (pRb) and induce the subsequent release of E2F transcription factors and expression of the genes required for the S phase. Cyclin-CDK complexes are regulated negatively by a family of kinase inhibitors
This study evaluated the relationship between GC responses, including GR expression and subsequent apoptosis, and p16, using the B-cell lymphoblast cell line NC-37.
The B-cell lymphoblast cell line NC-37 (ATCC number, CCL-214) was purchased from ATCC (Rockville, MD, USA). NC-37 cells were maintained in RPMI-1640 medium (Gibco BRL Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco BRL, Rockville, MD, USA) at 5% CO2 and 37℃ at saturated humidity.
For p16 siRNA transfection, a commercial kit was used (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Briefly, the following solutions were prepared: solution A contained 7 µL p16 siRNA (sc-36143) or control siRNA (sc-36869 or sc-37007) in 100 µL siRNA transfection medium (sc-36868). Solution B contained 6 µL siRNA transfection reagent (sc-29528) in 100 µL siRNA transfection medium.
Solution A was added to solution B directly, and the mixture was incubated for 30 min at room temperature. For each transfection, 0.8 mL siRNA transfection medium was added to each tube containing the siRNA and transfection reagent mixtures. In 6-well tissue plates, 3×105 cells were seeded per well and the mixtures were overlaid onto the cells. The cells were incubated for 6 h at 37℃ in a CO2 incubator, and then the transfection mixtures were removed and replaced with RPMI-1640 medium supplemented with 10% FBS and then incubated for an additional 6 h.
Western blotting was used to detect p16 protein after p16 siRNA transfection. Experiments were done for wild-type, control, and p16 siRNA-transfected NC-37 cells. The cells were collected by centrifugation, washed in phosphate-buffered saline (PBS), and lysed by the addition of SDS sample buffer (62.5 mM Tris-HCl [pH 6.8], 6% [w/v] SDS, 30% glycerol, 125 mM DTT, and 0.03% [w/v] bromophenol blue). Total cell samples were lysed and denatured by boiling for 5 min at 100℃. Equal amounts of protein from each sample were separated by 15% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked for 1 h with Tris-buffered saline containing 5% (w/v) milk and 0.1% Tween 20, then incubated with the primary rabbit monoclonal antibody for p16 (p16 INK4A Antibody; Cell Signaling Technology, Beverly, MA, USA) overnight at 4℃. The blots were washed with Tris-buffered saline containing Tween 20, incubated with the anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, USA) for 2 h, and developed using West-Zol TM plus (iNtRON Biotechnology, Seoul, Korea).
DX purchased from Sigma Chemical (St. Louis, MO, USA) was dissolved in dimethyl sulfoxide and added to the medium after p16 siRNA transfection. To study the effect of DX on p16 status, we added DX to samples containing wild-type, control, and p16 siRNA-transfected NC-37 cells. The final concentration of DX was adjusted to 100 nM. For measurements, cells were harvested 6, 12, 18, and 24 h after DX addition and then prepared for the next steps.
Cultured cells were washed twice in PBS containing 1% bovine serum albumin. Aliquots of 1×106 cells were fixed in paraformaldehyde at room temperature for 30 min, washed, and permeabil ized with 0.1% Triton X-100 in 0.1% citrate buffer for 5 min on ice. The cells were washed twice and incubated at 4℃ for 70 min with either FITC-conjugated anti-GR antibody (5E4; AbD Serotec, Oxford, UK) or FITC-conjugated isotype control (IgG1 AbD Serotec, Oxford, UK). Finally, the cells were washed and resuspended in PBS containing 1% bovine serum albumin and analyzed using a Coulter Elite flow cytometer (Beckman Coulter Inc., Fullerton, CA, USA).
To detect apoptosis, we performed double staining with FITC-Annexin V and propidium iodide (PI; R & D Systems, Minneapolis, MN, USA). First 1×106 cells were washed with PBS. FITC-Annexin V was diluted at a concentration of 1 mg/mL in binding buffer, and the cells were resuspended in 1 mL of this solution (prepared fresh each time). The resuspended cells were incubated for 10 min in the dark at room temperature, and then 0.1 mL PI solution was added to the cell suspensions before analysis to give a final concentration of 1 mg/mL. These cells were analyzed on using a Coulter Elite flow cytometer. Annexin V single-positive cells were regarded as early apoptotic cells, whereas Annexin V/PI double-positive cells were regarded as late apoptotic cells.
First 1×104 cells were suspended in 95 µL phenol red-free RPMI-1640 containing 0.1% FBS, then seeded into 96-well plates. DX was added to each plate at a concentration of 100 nM. After 6, 12, 18, and 24 h, 11 µL AB solution was added to the medium directly, resulting in a final concentration of 10%. As a negative control, AB was added to medium without cells. Then the plates were incubated for 4 h at 37℃. The absorbance of test and control wells was read at 570 and 595 nm with a standard spectrophotometer. The number of viable cells correlated with the magnitude of dye reduction and is expressed as a percentage of the AB reduction compared to control AB.
All statistical analyses were conducted using SPSS 13.0 (SPSS, Chicago, IL, USA). The results were expressed as the mean±standard deviation, and the differences between the cell groups at each time point were analyzed using the Mann-Whitney
To determine whether p16 affects GR regulation and the apoptosis of lymphoblast cells, we generated p16 siRNA-transfected NC-37 cells that did not express p16. First we used the florescence-expressing control siRNA to confirm p16 siRNA transfection. When more than 50% of the control siRNA-transfected NC-37 cells had fluoresced, Western blot analysis was performed to detect p16. The wild-type and control NC-37 cells expressed p16 in the immunoblot analysis, whereas the p16 siRNA-transfected NC-37 did not (
We evaluated time-dependent GR expression 0, 6, 12, 18, and 24 h after treatment with DX for control (p16+ NC-37) and p16 siRNA-transfected (p16- NC-37) cells. The GR levels were determined by flow cytometer after intracellular GR staining at each time point. After DX treatment, GR expression began to increase after 6 h, reached a peak at 18 h, and decreased sharply by 24 h (
We assessed the time-dependent apoptotic changes after DX treatment using Annexin V/PI staining of cells by flow cytometry. After the DX treatment, both p16+ and p16- NC-37 cells showed a marked initial increase in Annexin V-stained cells (the early apoptotic cells) at 6 h, followed by a decrease at 24 h (both
The late apoptotic cells (double-positive cells) in p16- NC-37 cells increased through 6~18 h and reached a maximum at 18 h, and >50% of the cells were apoptotic. In contrast, the late apoptotic cells increased in a time-dependent manner over 24 h in p16- NC-37 cells. Overall, p16+ NC-37 cells was more susceptible to DX-induced late apoptosis than p16- NC-37 cells, and the result at 18 h was significant (
We assessed time-dependent cell viability using the AB assay. AB is a redox indicator that produces a colorimetric change in the fluorescent signal in response to metabolic activity. Within 12 h, the cell viability between p16+ and p16- NC-37 cells was not significantly different. At 18 and 24 h, the cell viability was reduced compared to the value at 12 h in both groups (
Clinically, GC resistance indicates a poor prognosis in ALL treatment
The inactivation of p16 has been confirmed to varying degrees in T-cell leukemias and other hematologic malignancies
Few studies have examined the relationship between GC responsiveness and p16. In this study, we evaluated the time course of GC-induced GR expression changes and the effect of p16 on GC-induced apoptosis using p16 siRNA transfection of a B-cell lymphoblast cell line, NC-37 cells. We found a pattern of intracytoplasmic GR expression after DX treatment within 24 h. That is, the initial GR levels peaked at 18 h, followed by a sudden decrease at 24 h in p16+ and p16- NC-37 cells; the former tended to show higher expression rates (
Combined, our results suggest that p16 is positively correlated with GR expression and GC-induced late apoptosis. Further effort is necessary to identify additional genetic changes in cell cycle regulators to complement these findings. Studies of p16 and GC responsiveness may lead to new treatment modalities, such as a combination of GC with substances mimicking p16 function, including CDK4- or CDK6-inhibiting peptides, for hematologic malignancies that do not express p16
In conclusion, although p16 has a role in GR expression and apoptosis induced by GC, it remains to be seen whether the GC-induced apoptosis mechanism can interact with the other signaling events associated with the p16 gene operating during such malignant changes. This observation might have important implications for cancer therapy.
Western blot analysis of p16 compared with β-actin in NC-37 cells. Wild-type, control siRNA-transfected, and p16 siRNA-transfected NC-37 cells were immunoblotted with p16 antibody. The p16 siRNA-transfected NC-37 cells did not express p16 protein. The bar graphs express the ratio of p16 to β-actin calculated from densitometry measurements. *
Cytoplasmic glucocorticoid receptor (GR) expression levels as measured by flow cytometry. Time-dependent changes in GR expression after dexamethasone (DX) treatment are shown for control and p16 siRNA-transfected NC-37 (A) along with the results of flow cytometry (B). GR expression peaked at 18 h and decreased sharply at 24 h. The control NC-37 cells expressed higher glucocorticoid receptor (GR) levels compared to the p16 siRNA-transfected NC-37 cells at 18 h. *
Apoptotic cells stained with annexin V and propidium iodide (PI) as assessed by flow cytometry in both groups. Annexin V single-positive cells were regarded as early apoptotic cells (A) and double-positive cells were regarded as late apoptotic cells (B). There were no statistical differences between the 2 groups for early apoptosis (A). Late apoptotic cells increased in a time-dependent manner and peaked at 18 h in the control NC-37 cells. *
Alamar blue (AB) assay estimating time-dependent cell viability. Cell viability decreased after dexamethasone (DX) treatment. After 12 h, viability of the control NC-37 cells decreased more rapidly than that of the p16 siRNA-transfected NC-37 cells. *