The present study was approved by the Ethics Committee of the Medical University of Lodz (Lodz, Poland; approval no. RNN/143/10/KE). Informed consent was provided for the use of patient data in this study. The first sample was collected in July 2012 during stable disease when the patient was 22 years old and the second sample was taken 18 months later in January 2014, during progressive disease. The white blood cell count was 120,000 cells/µl in 2012 and 657,000 cells/µl in 2014. CLL diagnosis of this patient was performed in Hematology Department of Medical University of Lodz at the Copernicus Memorial Hospital (Lodz, Poland) according to standard clinical, immunological and cytological criteria from the International Workshop on Chronic Lymphocytic Leukemia (1,22,23).

Following blood sample analysis, fludarabine + cyclophosphamide administration was commenced, according to standard pharmacological conditions (22). The patient received fludarabine (Teva Pharmaceutical Industries, Ltd., Petah Tikva, Israel) at a dose of 25 mg/m² combined with cyclophosphamide (Baxter International, Inc., Deerfield, IL, USA) at 250 mg/m² for 3 days intravenously (FC regimen), every 28 days for 6 cycles. Clinical response to the treatment following 6 cycles of drug administration was evaluated as previously described (22,24). Following this treatment, the patient achieved complete remission.

PBMCs were isolated from the two blood samples using Histopaque-1077 (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) according to the manufacturer's instructions. The obtained cells were washed with PBS and cultured at a final density of 3×10⁶ cells/ml in RPMI-1640 medium (Cytogen GmbH, Zgierz, Poland) containing 10% fetal calf serum (Biowest, Nuaillé, France), 2 mM L-glutamine (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), 100 U/ml penicillin and 100 µg/ml streptomycin (Biowest). The leukemic PBMCs were subsequently incubated with anticancer agents. Cells were incubated with CM, FM, RCM, Rit or RK as described previously (25,26). Cladribine was supplemented in dose of 50 µg/ml, mafosfamide at 1 µg/ml, Rit at 20 µg/ml, fludarabine at 20 µM and kinetin riboside at 20 µM. A group not treated with any agents was used as a control. The leukemic cells were incubated without any agents (control) or treated with these anticancer agents for 48 h at 37°C with 5% CO₂.

Cladribine (Biodrybin), fludarabine and rituximab were obtained from the Institute of Biotechnology and Antibiotics (Warsaw, Poland), fludarabine was obtained from Teva Pharmaceutical Industries, Ltd. and Rit was obtained from Roche (Basel, Switzerland). Mafosfamide was obtained from Baxter Oncology GmbH (Frankfurt, Germany) or purchased from Niomech IIT GmbH (Bielefeld, Germany). The kinetin riboside was obtained from the Institute of Bioorganic Chemistry (Polish Academy of Science, Poznan, Poland).

The leukemic cells were incubated in culture medium, with or without (the control) drug treatment. The fractions of viable and apoptotic cells following 48 h of exposure were determined by Vybrant Apoptosis assay kit #4 (Molecular Probes; Thermo Fisher Scientific, Inc., Waltham, MA, USA), as described previously (15).

Following a 48 h incubation period, the control and drug-treated CLL cells were rinsed with PBS and homogenized in isotonic sucrose solution [5 mM MgCl₂, 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.4), protease inhibitors] as described previously (26). The homogenate was then centrifuged at 1,000 × g for 10 min to obtain the nuclear fraction. The leukemic cells were subsequently lysed in a buffer containing 10 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1% Triton X-100, 2 mM MgCl₂, 0.1 M dithiothreitol and a mixture of protease inhibitors, as described previously (26). The protein content was measured by standard Lowry method.

The protein samples (40 µg/lane) were separated by SDS-PAGE on an 8% gel and blotted onto an Immobilon-P polyvinylidene fluoride membrane. Protein loading and transfer were confirmed by Ponceau S staining. The membranes were blocked with 5% non-fat powdered milk dissolved in TBS [10 mM Tris-HCl (pH 7.5) and 150 mM NaCl] for 1 h at room temperature. Subsequent to washing in TBS containing 0.05% Tween-20, the blots were incubated with a primary antibody directed against PARP-1 (cat. no. sc-7150; dilution, 1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), myeloid leukemia cell differentiation protein (Mcl-1; dilution, 1:3,000) or B-cell lymphoma 2 (Bcl-2; dilution, 1:2,000) produced by Santa Cruz Biotechnology, Inc. An alkaline phosphatase-conjugated secondary antibody was then used, as previously described (26).

Nuclear fraction preparations of the control and treated cells were prepared for calorimetric tests according to the protocol used by Almagor and Cole (27). The nuclear fraction samples were transferred into pans and hermetically sealed. Calorimetric experiments were performed using a TG-DSC 111 calorimeter (Setaram, Caluire, France). A temperature of 20–120°C and a scanning rate of 5°C/min (0.083°C/s) was used, as previously reported (28,29).

The amount of apoptotic and necrotic cells up to 48 h following drug exposure were monitored using a fluorescence microscope (IX70; Olympus Corporation, Tokyo, Japan; magnification, ×400). The cells were suspended in PBS at a density of ~1×10⁶ cells/ml, incubated for ~20 min (room temperature, in the dark) with YO-PRO-1 and propidium iodide (PI), and transferred onto microscopic slides. The YO-PRO-1 fluorescent dye selectively labels apoptotic cells green. PI stains late apoptotic and necrotic cells red. The stained cells were classified as apoptotic and necrotic, as previously described (30).

The results of the cell viability assay, DSC, immunoblotting and proteolytic degradation of the apoptotic marker PARP-1 highlighted the differences between the classic stable and progressive forms of the disease. The most marked changes were observed in the fractions of living and apoptotic cells (Fig. 1), as well as in the nuclear fraction profiles analyzed by DSC (Fig. 2) and PARP-1 expression/proteolytic degradation (Fig. 3). Additionally, a reduced number of apoptotic leukemic cells was observed during progressive disease (Fig. 1B). In addition, variation was observed in the proteolytic degradation of PARP-1 in leukemic cells incubated with RK, in the stable (Fig. 3A) and progressive forms of CLL (Fig. 3B). These results demonstrate the differences in CLL cell sensitivity to anticancer agents between the stable and progressive forms of the disease.

The combination of tests useful in personalized therapy for CLL (15) may be valuable in monitoring aspects of CLL progression, particularly the diversity in potential of leukemic cells to induce apoptosis during disease development. In addition, these tests could track the changes in chromatin structure that occur when cell proliferation is induced, which is indicative of the progressive form of the disease (Fig. 2B). These results contrast with the cell features identified during the characteristic stable form of the disease (Figs. 1A–3A).

A comparative analysis was performed between the blood samples obtained during stable and progressive CLL. The comparison of the results from the four tests performed revealed that the stable form of the disease exhibited the typical features of apoptosis induction, whereas the progressive disease revealed a high proliferative index and elevated leukocytosis score.

In the stable form of the disease, all anticancer agents tested induced a higher level of PBMC apoptosis compared with the control group (Fig. 1A). However, when the same drugs were applied to the leukemic PBMCs from progressive CLL, a reduced level of apoptosis was observed (Fig. 1B). Changes in corresponding cell viability were noted subsequent to incubation with CM, FM and RCM. The PBMCs exhibited a lack of activity when cultured with Rit. Notably, RK demonstrated the greatest apoptotic induction in stable (Figs. 1A and 3A) and progressive leukemic cells (Figs. 1B and 3B). The DSC profiles of the nuclear fraction confirmed the presence of diverse changes in CLL chromatin conformation, resulting from exposure to the tested agents (Fig. 2). Considerable differences in leukemic cell reactivity were observed in cell samples incubated with Rit, with the cells appearing resistant to Rit in the stable form of the disease. Previous studies have reported that in progressive CLL, additional thermal transition occurs at ~95±5°C (15,19,20,26,28,29), which decreases in the case of the apoptotic activity. The presence of a thermal transition at 95°C following CLL incubation with ≥1 of the agents indicates that the leukemic cells in the stable form of the disease are resistant to Rit.

Proteolytic degradation of the apoptotic marker PARP-1 was observed in cells undergoing drug-induced apoptosis (Fig. 3). However, although PARP-1 was cleaved when resting leukemic cells were incubated with RK, this was not the case in the progressive form of the disease. In addition, PARP-1 was not cleaved in cells following exposure to Rit, suggesting that Rit is not effective, particularly in the stable form of the disease. The proapoptotic degradation of the antiapoptotic proteins Mcl-1 and Bcl-2 corresponded with other results of the present study, demonstrating induction of apoptosis. Notably, in the progressive form of CLL, a decreased apoptosis level was found to be associated with Bcl-2 expression.

PBMCs from the progressive form of CLL were analyzed for apoptosis and necrosis cells (Fig. 1C), in order to investigate which anticancer drug(s) were inducers of apoptosis in CLL cells. In the studied samples, from the CLL patient, CM and FM demonstrated strong activity as apoptotic inducers in leukemic PBMCs, indicating that they may be effective in the treatment of progressive CLL. In contrast, the most effective drug for the treatment of the stable form of disease in the patient studied was RK (Figs. 1A–3A), a novel purine analog with high apoptosis potential.