High Concentrations of Anaerobic Adenosine-Triphosphate May Impair Apoptosis In Malignant B Cells From Patients With Chronic Lymphocytic Leukemia

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High concentrations of anaerobic ATP implicated in aborted apoptosis from CLL ¹Udristioiu Aurelian, ²Cristina Florescu, ³Manuela Anda Radu-Popescu, 4 Manole Cojocaru ¹Emergency County Hospital TARGU-JIU,.Progresului Street No.18,Gorj, Postal Code:Zip 210218,Romania, ²Central Laboraotry Synevo, Clinical Trial, District 1, Bucharest, Romania ³UMF Carol Davila, Medicine Faculty, Department of Farmacy, Bucharest, Romania 4Titu Maiorescu University, Faculty of Medicine, Physiology Department, Bucharest, Romania ABSTRACT Introduction Apoptosis is an energy related process, in contrast with necrosis that occurs in the absence of adenosine-triphosphate (ATP). The main objective of the study was to assess intracellular ATP concentrations in B lymphocytes from patients with chronic lymphocytic leukemia (CLL), in comparison with ATP concentrations from B and T cells from patients with malignant diseases or non-malignant diseases, with allergic conditions. Material and method Using a Luminometer LKB analyzer, on principle of bioluminescence, we examined 75 patients (50 males and 25 females) with the following diagnoses: 25 patients (mean age 55 ± 0.5)) with allergic diseases (chronic allergic rhinitis, allergic dermatitis, chronic allergic asthma), 25 patients (mean age 58 ±1.8) with nonhematopoietc malignant diseases (lung cancer, bone metastasis) and 25 patients with confirmed diagnosis of CLL. Control group consisted of 120 healthy patients (mean ages 40.02 ±12.01). Results In the healthy male and female subjects, the mean concentration of ATP in 1 x 106 lymphocytes /ml of normal peripheral blood, T lymphocytes had was 1.39 µM ATP and in B lymphocytes the concentration was 0.35 µM ATP/ml [SD = 0.41, p= 0.030]. The mean concentration of ATP in 1 x 106 activated peripheral blood T lymphocytes from patients with allergic diseases was 3.12 µM ATP/ml and in B lymphocytes the mean concentration was 0.79 µM ATP/ml [SD= 0.56, p = 0.034]. From patients with malignant diseases, the concentration of ATP in 1 x 106 activated peripheral blood T lymphocytes/ml had a mean value of 3.06 µM ATP and the mean concentration of ATP in activated 1 x 106 peripheral blood B lymphocytes/ml was 0.17 µM ATP [SD= 0.45, p = 0.05]. The mean concentration of ATP in 1 x 106 activated peripheral blood malignant CLL B lymphocytes/ml was 4.33 µM ATP but was only 0.09 µM ATP [SD= 1.5, p< 0.05] in T lymphocytes from these patients. A strong correlation was observed between the concentration of ATP of T lymphocytes from patients with malignant diseases and ATP concentration of B lymphocytes from samples of patients with CLL (r= 0.99) and a good correlation was observed between T lymphocytes (Th) from allergic diseases and T lymphocytes (Ts) from malignant diseases (r = 0.94). Conclusion Blocked apoptosis from malignant diseases may be due to high ATP concentration originating from anaerobic metabolism. The difference of energy between anaerobic ATP in B lymphocytes from CLL and aerobic ATP in T lymphocytes from normal status in value of 2.68 µM ATP, as an energetic transfer between B and T cells, initiates carcinogenesis by suppression of anti oncogene proteins, especially p53 protein. Further studies are necessary to detect to patients with high concentrations of ATP and the mutations, translocations or deletions of the p53 gene, which is located on chromosome 17, using FISH technology. Abbreviations

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Materials and methods The measurement of ATP was made by standard principle of bioluminescence on automatic analyzer LKB, using ATP monitoring reagent, ATP Standard, (106 Mol ATP/ ml), buffer solution Tris-EDTA, TCA-EDTA lyse. All results were statistically analyzed by Excel program. T lymphocytes were obtained from the peripheral blood of healthy individual and from hospitalized patients with selected diseases from the departments of Internal Medicine and Oncology. Patient cohort was a total of 75 patients (50 males and 25 females) consisting of the following groups were examined: 25 patients (mean ages 55 ± 0.5)), with allergic diseases (allergic rhinitis, allergic dermatitis , chronic allergic asthma), 25 patients( mean ages 58 ± 1.8 ) with nonhematopoietc malignant diseases ( lung cancer, bone metastasis) and 25 patients with confirmed hematopoietic malignant disorders, in diagnosis of CLL. In addition, 120 blood samples from apparently healthy donors (mean ages 40.02 ±12.01) were also examined. The May Grunwald-Giemsa method was used to determine the microscopic appearance of peripheral blood lymphocytes from patients with incipient CLL disease, who had not had any treatment or hematopoietic stem cell transplantation (HSCT).Morphologically, the cells resembled normal mature clonal B cells arrested in the B- cell differentiation pathway intermediate between pre-B cells and mature B cells lymphocytes although slightly larger, and more fragile when smeared onto a glass slide(smudge cells)Lymphocytes were separated from peripheral blood by centrifugation in a Ficoll gradient for 20 minutes at 2000 rcf/ minute. Lysis of lymphocytes with TCA-EDTA was performed after cell counts were adjusted to 1 x 106 lymphocytes /ml of plasma. An initial panel of monoclonal antibodies was used to immune-phenotype T cells and their subgroups and B cells and their subgroups. Immune-cytochemical detection of antigen was made using the ABC-AP, APAAP method. Activated B lymphocytes were defined as CD19 + cells, CD20, CD21, and CD23 and or CD138 surface marker. Activated T cells were identified with CD7, CD5, CD3, CD2, CD4, CD8 and CD45RO. The phenotype suggestive of B-CLL or monoclonal B cell lymphocytes was approximately equal numbers of CD5+ and CD19+ lymphocytes. For monitoring diagnosis and prognosis of CLL, the samples were sent to the National Institute of Reference Oncology, Fundeni. Expected values to cytogenetic results [7] will the deletion13q chromosome that can occurs in more than 50% of patients and trisomy 12, which can occurs in about 60 of patients. Individual13q14 abnormalities have a relatively benign disease that usually manifests as stable or slowly progressive isolated lymphocytosis. Deletion in the short arm of chromosome 17 is associated with rapid progression, short remission, and decreased overall survival in chronic CLL. The 17p13 deletions are associated with loss of function of the tumor suppressor gene p53 and deletions of bands 11q22-q23, associated with extensive lymph node, involvement aggressive disease and shorter survival. In the differential diagnosis of CLL we included: -Hairy cell leukemia, which is moderately positive for surface membrane immunoglobulins of multiple heavy-chain classes and is typically negative for CD5 and CD21. -CLL should also be distinguished from pro-lymphocytic leukemia, in which more than 65% of the cells are morphologically less mature pro-lymphocytes. - Proymphocytic leukemia has a typical phenotype that is positive for CD19, CD20, and surface membrane immunoglobulin but one half will be negative for CD5. Large granular lymphocytic leukemia has a natural killer (NK) cell phenotype (CD2, CD16, CD56) or a T-cell immune-type (CD2, CD3, CD8 -Pattern of positively for CD19, CD20, and the T-cell antigen CD5 is shared only by mantle cell lymphoma and these cells generally do not express CD23. Results:

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