Potential protein markers in diagnosis and treatment of B-CLL

M. Rogalinska MSca., J. Blonski b, M.D., Ph.D., M. Hanausek c Ph.D., T. Robak b M.D., Ph.D., Z.M. Kilianska a Ph.D.

a Department of Cytobiochemistry, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland; b Department of Hematology, Medical University of Lodz, Pabianicka 62, 93-513 Lodz, Poland, c AMC Cancer Research Center, 1600 Pierce Street, Denver CO 80214, USA

AIM: Chronic lymphocytic leukemia (CLL) is a hematological neoplasm characterized by a uncontrolled expansion of low proliferative clone of B-cells that are not able to differentiate into mature cells and can not enter on apoptotic path. The remarkable variability in clinical presentation, course and prognosis was a challenge in research on cancer-specific associated proteins. Our previous data revealed that two electrophoretically-specific nuclear non-histone proteins with Mr of 44/46 and 38/39 kDa (p44/46 and p38/39) were identify as a leukemia-specific polypeptides. The aim of present study was to employ a new approach that could compare the composition of nuclear proteins from normal and B-CLL mononuclear cells. It is accepted that in this type of leukemia apoptosis is blocked. Recently, we have performed our study on apoptotic markers in lymphocytes of drug-treated patients. Using a few apoptotic markers (PARP, Bcl-2, lamin B) we tried to find more effective type of B-CLL patients chemotherapy. METHODS: Peripheral blood samples were obtained from B-CLL patients. Nuclei or/and homogenate and post-nuclear fraction were isolated by the sucrose method. In the study we used SDS-PAGE for analysis of protein specificity. The rabbit antisera raised against p44/46 and p38/39 were used in Western blot and ELISA detection of these leukemia-associated antigens. The efficacy of treatment based on activation of lymphocyte-apoptosis with three drug systems (2-chlorodeoxyadenosine 2CdA, [C]; 2CdA + cyclophosphamide [CC]; 2CdA + mitoxantrone + cyclophosphamide [CMC] was analyzed using Western blot technique. RESULTS. The diversities in protein composition of nuclear, mitochondrial, microsomal, and cytosolic fractions from normal and B-CLL mononuclear cells was reported by our group [Leukemia Res., 23(1999):833-841]. Our special interest was focused on two electrophoretically-specific non-histone proteins, i.e., p44/46 and p38/39. The Western blot analysis indicated that both polypeptides are specifically expressed in leukemia mononuclear cells but not in those isolated from blood of healthy donors. Very recent study increased a number of tested blood probes and also introduced a simple quantitative ELISA test for the screening of potential markers for B-CLL cells. We have also expanded study on induction of apoptosis in patients lymphocytes after different drug(s) treatment. Preliminary studies indicated that cleavage profiles of PARP and lamin B and level of Bcl-2 expression during three studied types of drug treatment are different in comparison to untreated patients. These differences seems to be type of drug- and patient's personal sensitivity-dependent. CONCLUSIONS. Neoplastic transformation of lymphocytes is accompanied by specific expression of non-histone proteins with Mr of 44/46 and 38/39 kDa; 2CdA alone or in combination with other drugs induce apoptosis in B-CLL cells; three drugs regimen seems to be the most effective in activation of apoptosis in studied cells.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Predictive Markers.