Materials and methods (Supplementary Information)
Cell lines, culture conditions, and reagents. CEM-C7H2 (1) is a subclone of the CCRFCEM-C7 cell line (2). Generation and analysis of the p16INK4A-expressing clones 6E2/p16 and
1D2/p16 have been described before (3). All cells were maintained in RPMI 1640 containing
10% fetal calf serum (FCS; Gibco BRL, GB), 100 U/ml penicillin, 100 µg/ml streptomycin
and 2 mM L-glutamine (Gibco BRL, GB) at 5% CO2 and 37°C in saturated humidity.
Northern blot analysis. Total RNA was extracted from 5 x 106 cells with TriReagentTM
(LPS, Moonachie, NJ, USA). Twenty µg RNA were separated by electrophoresis and blotted
overnight onto ZetabindTM nylon-membranes (Cuno, Meridien, CO, USA) according to
standard protocols. RNA was cross-linked to the membranes by UV light. Filters were
hybridized at 65°C over night with
32
P-dATP-labeled, heat-denatured full-length probes
specific for E2F-1, dehydrofolate reductase (DHFR), ornithine decarboxylase (ODC),
prothymosine  (PTM), c-Myc, glycerinaldehyde-dehydrogenase (GAPDH) and 18S-RNA,
respectively. The washed blots were exposed to Agfa Curix X-ray films. Between
hybridizations, the blots were stripped by boiling in 0.1% SDS.
Determination of cell numbers, volume and apoptosis. Cell numbers and cell volume were
measured by a Casy TT cell counter (Schärfe Systems, Germany). Apoptosis was quantified
by nuclear staining with propidium iodide (PI) in concert with forward/sideward scatter
analysis (4) in a Beckton Dickinson FACScan. Nuclei in the sub-G1 marker window were
considered to represent apoptotic cells.
Immunoprecipitation and CDK kinase activity assays. For detection of CDK-bound
proteins and CDK-kinase activity, 2 x 107 cells were lysed in IP-buffer (50 mM HEPES pH
7.5, 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 25 mM -glycerophosphate, 10%
glycerol, 1 mM PMSF, 1 mM DDT, 50 mM NaF, 5 mM Na3VO4, 50 µg/ml aprotinin, 10
µg/ml leupeptin) for 30 min on ice. Lysates were incubated at 4°C over night with 1 µg
polyclonal rabbit antisera (BD-Pharmingen, Germany) directed against CDK2, CDK4 or
CDK6 and precipitated by 2,5 µg PansorbinTM (Calbiochem, La Jolla, CA, USA). The pellets
were either used for immunoblotting or incubated in kinase buffer (25 mM HEPES pH 7.5, 2
mM MnCl2, 20 mM MgCl2, 1 mM NaF, 10 mM -glycerophosphate, 1 mM Na3VO4)
containing 20 µM ATP with 10 µCi [32P]ATP plus 5 µg histone H1 (Sigma, Austria) at 30°C
for 30 min. Reactions were stopped by adding loading-buffer and boiling. Labeled proteins
were separated on SDS polyacrylamide gels that were afterwards subjected to
autoradiography in a Packard Instant Imager.
Immunoblotting. Cells were lysed on ice in CelLytic-M buffer (Sigma, Austria). Samples
were separated by SDS-PAGE on 7.5-15% polyacrylamide gels and transferred to
nitrocellulose membranes by a Hoeffer semi-dry transfer apparatus. The membranes were
blocked with TRIS-buffered saline (TBS) containing 1% Tween20 and 5% nonfat dry milk,
incubated with primary antibodies specific for human Cyclin D3, Cyclin E, Cyclin A, p27Kip1,
CDK2, CDK4, CDK6, E2F-1, c-Myc, p107, Mad, Max, pRB (BD-Pharmingen, Germany),
and -Tubulin (Oncogene Research, MA, USA) over night and detected with horseradishperoxidase-conjugated secondary antibodies (Amersham, UK). The blots were developed with
ECL (Amersham, UK).
Immunofluorescence and mitochondrial staining. 2 x 104 cells were centrifuged onto
slides, fixed with ice-cold 70% ethanol. The cells were incubated with a monoclonal p16 INK4A
antibody (BD-Pharmingen, Germany) diluted in PBS/1% BSA for 30 min, washed and
incubated with a FITC-conjugated antibody (DakoCytomation, Denmark). H&E staining was
performed with Meyer’s hemalaun-solution and 1% eosine. For quantification of surface
marker expression 1 x 106 cells were incubated with FITC-conjugated antibodies directed
against CD3 and CD4 (BD-Pharmingen, Germany) diluted in PBS/1%BSA and analyzed in a
Beckton Dickinson FACScan. For mitochondrial staining, 1 x 105 cells were incubated in PBS
containing 30 nM MitoTracker red (Molecular Probes, Netherlands) for 10 min at 37°C,
washed and measured in a FACScan.
Telomeric Repeat Amplification Protocol (TRAP) assay. For determination of telomerase
activity, 2.5 x 105 cells were lysed and analyzed using a PCR-based TRAP protocol. The PCR
amplification products were separated on 15% nondenaturating polyacrylamid gels and
stained with SYBR Green (5).
Determination of protein-synthesis and ATP content. For determination of protein
synthesis, equal numbers of cells were washed in methionine-free RPMI 1640, incubated for
30 min in RPMI 1640 containing 10% dialyzed FCS and then cultured for another 3 hours in
the presence of 50 µCi
35
S-methionine. Incorporated radioactivity was measured in a
scintillation counter. Cellular ATP was measured according to the manufacturer’s instructions
(ATP-determination Kit, Molecular Probes, Netherlands). Briefly, 2x105 cells were lysed and
mixed with 100 µl reaction solution containing luciferin and luciferase. After 10 minutes
incubation at room temperature, luciferase activity was assessed in a standard luminometer
(Berthold Technologies).
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