ALBERT

Description: Although B-cell chronic lymphocytic leukemia (B-CLL) clones with unmutated IGHV genes (U-CLL) exhibit greater telomerase activity than those with mutated IGHV genes (M-CLL), the extent to which B-cell receptor (BCR) triggering contributes to telomerase up-regulation is not known. Therefore, we studied the effect of BCR stimulation on modulating telomerase activity. The multivalent BCR ligand, dextran conjugated anti-μ mAb HB57 (HB57-dex), increased telomerase activity and promoted cell survival and proliferation preferentially in U-CLL cases, whereas the PI3K/Akt inhibitor LY294002 blocked HB57-dex induced telomerase activation. Although both U-CLL and M-CLL clones exhibited similar membrane proximal signaling responses to HB57-dex, telomerase activity and cell proliferation, when inducible in M-CLL, differed. B-CLL cells stimulated using bivalent F(ab′)2 -goat anti-μ antibody (goat anti-μ) exhibited higher membrane proximal response in U-CLL than M-CLL cells, whereas telomerase activity, cell survival, and proliferation were induced to lower levels than those induced by HB57-dex. In normal B lymphocytes, HB57-dex induced less protein phosphorylation but more cell proliferation and survival than goat anti-μ. Although both anti-BCR stimuli induced comparable telomerase activity, normal CD5+ B cells preferentially exhibited higher hTERT positivity than their CD5− counterparts. These findings provide an understanding of how BCR-mediated signals impact telomerase modulation in IGHV mutation-based subgroups of B-CLL and normal B cells.

Description: An antigen-independent process that takes place in the bone marrow (BM) leads to the birth of B cells from bone marrow precursors. Cross-talk between components of the microenvironment of BM or secondary lymphoid compartment(s), eg. spleen (SP), nurtures the subsequent evolution of B cells and governs in large part the natural history of normal B cells and B cell malignancies including chronic lymphocytic leukemia (B-CLL). Interaction of B cells with stromal elements confers upon them features enabling their transient sequestration, proliferation and extended survival. In this report we have compared the characteristics of clonal B-CLL cells obtained from paired specimens (peripheral blood, PB with corresponding BM or SP obtained within an interval of less than 1 month of each other) from 17 individual untreated B-CLL patients. These cells were tested by surface immunofluorescence and flow cytometry for their expression of a panel of chemokine receptors (CCR −1, −2, −4, and −7 and CXCR−1, −2, −3, and −4) and markers of cellular activation (CD23, CD62L, CD69, CD71 and HLA-DR). The relative age of the B cells (telomere length) and their ability to maintain telomere length (telomerase activity) were studied in paired BM/SP and PB samples from 15 of these cases. Although PB-, BM- and SP-derived B cells expressed activation markers, specifically, the percentages of cells expressing ZAP-70 and Ki-67 were significantly higher in BM- and SP-derived B cells than those expressed by corresponding PB-derived B cells (p

Description: Chronic lymphocytic leukemia (CLL) cells are thought to have diminished cell-cycling capacity, a view challenged by their phenotypic resemblance to activated human B lymphocytes. The present study addresses the cell-cycling status of CLL cells, focusing on those leukemic cells expressing CD38, a molecule involved in signaling and activation that also serves as a prognostic marker in this disease. CD38+ and CD38− members of individual CLL clones were analyzed for coexpression of molecules associated with cellular activation (CD27, CD62L, and CD69), cell-cycle entry (Ki-67), signaling (ZAP-70), and protection from apoptosis (telomerase and Bcl-2). Regardless of the size of the CD38+ fraction within a CLL clone, CD38+ subclones are markedly enriched for expression of Ki-67, ZAP-70, human telomerase reverse transcriptase, and telomerase activity. Although the percentage of cells (approximately 2%) entering the cell cycle as defined by Ki-67 expression is small, the absolute number within a clone can be sizeable and is contained primarily within the CD38+ fraction. Despite these activation/proliferation differences, both CD38+ and CD38− fractions have similar telomere lengths, suggesting that CD38 expression is dynamic and transient. These findings may help explain why high percentages of CD38+ cells within clones are associated with poor clinical outcome.

Description: Telomere shortening is a consequence of repetitive clonal replication and leads to clonal deletion unless DNA extension and repair occur. All tumors must circumvent this problem by up-regulating mechanisms that lead to chromosomal lengthening. Two mechanisms have been identified that maintain chromosome ends- telomerase that does so by reverse transcription and alternative lengthening of telomeres (ALT) that occurs by homologous recombination. The latter function is characterized by the presence of promyelocytic leukemia protein-associated nuclear bodies (PML-NBs) and the presence of PML-NB is used to mark cells that use this process. B cell Chronic lymphocytic leukemia (B-CLL) cells with unmutated Ig V genes have shorter mean telomere lengths compared with those exhibiting mutated Ig V genes. In addition, cells with unmutated Ig V genes demonstrate more telomerase activity than their mutated counterparts. The mutated cases show long and heterogeneously elongated telomeres in spite of the absence, in most cases, of detectable telomerase activity. Therefore we determined whether the ALT pathway plays a role in telomere maintenance in B-CLL, using a monoclonal anti-PML antibody and a flow-cytometric assay for assessment of PML protein. Telomerase-expressing Jurkat T cells and murine fibroblasts-L cells served as negative controls for PML staining, whereas the ALT positive Osteosarcoma cell line U2-OS served as a positive control. In a cohort of 20 B-CLL cases, PML protein was detected in all cases regardless of Ig V mutation status. In addition, a similar percentage of cells within the clones contained PML (10 - 90% of the members of unmutated clones and 11–96% of mutated clones), whereas peripheral blood B cells from 6/6 elderly normal donors did not show any PML staining. PML expression was compared with telomere length and telomerase activity in the same cases. The percentage of cells showing PML expression inversely correlated with telomerase activity (r= −0.58; p=0.029). Although in most published reports telomere maintenance by ALT occurs in the absence of telomerase activity, we found ALT (as suggested by PML positive cells) in cells with telomerase activity (detected by the standard TRAP assay). Thus, B-CLL cases can express PML bodies and some B-CLL cells can contain both PML-NB and express telomerase activity. These findings suggest that B-CLL cells can use two distinct mechanisms to assure telomere maintenance and perpetuate clonal survival and expansion.

Description: B-cell chronic lymphocytic leukemia (B-CLL) has been considered for years as a slowly progressive invasion of phenotypically homogeneous, monoclonal B cells that divide rarely and accumulate because of a primary apoptotic defect. Recent studies are clarifying that the leukemic clone can be comprised of subpopulations of cells expressing different surface markers suggestive of intraclonal diversity. Indeed, it is now widely accepted that activation signals (i.e., antigenic stimulation, interaction with accessory cells, cytokines) can push B-CLL cells into the cell cycle or rescue them from apoptosis. Unanswered questions are whether all or only a subset of cells in the clone participates in these proliferative events and to what extent those cells that have divided can be identified. We have tried to answer this by labeling those cells that divide in vivo with the non-radioactive stable isotope deuterium (2H) in the form of deuterated water. Nine B-CLL patients with leukemic clones comprising 1.7 – 97.8% CD38+ cells drank daily, for 12 weeks, an aliquot of 2H2O. CD19+CD5+CD38+ and CD19+CD5+CD38− cells were isolated from PBMC drawn after 8 weeks (labeling period) and 24 weeks (washout period) and the incorporation of 2H into equal amounts of genomic DNA was measured by gas chromatography/mass spectrometry. At week 8, the mean 2H enrichment in the CD38+ population was higher than in the CD38− fraction (0.862 ± 0.24 vs. 0.445 ± 0.13; P = 0.034). Enhanced 2H enrichment in the CD38+ population was detected in every patient with CD38+/CD38− ratios ranging from 1.15 to 5.58. In 4 patients, the CD38+/CD38− ratio was above 2.0. In contrast, at week 24, 12 weeks after the cessation of 2H2O intake, 2H enrichment in DNA in the CD38+ vs. the CD38− population had changed. In 7 of 9 patients, the CD38+/CD38− enhancement ratios diminished, remaining essentially constant for the other 2 patients. The difference of CD38+/CD38− mean ratios at week 8 and week 24 was significant (2.254 ± 0.12 vs. 1.124 ± 0.13; P = 0.035). Moreover, the incorporation of 2H into CD38+ cells fell in 6 of 9 patients, with reciprocal increases in label incorporation in the CD38− cells for 8 of 9 patients. Thus, the differences in 2H incorporation into DNA at weeks 8 and 24 suggest either that the life spans of the two subpopulations differ (i.e., CD38+ cells may be shorter lived) or that expression of CD38 may be a dynamic process in vivo changing from positive to negative in individual cells over time after replication. These in vivo findings indicate that, within the same B-CLL patient, the leukemic subpopulation that expresses CD38 contains more cells that have recently undergone cell division than the CD38− subpopulation. This may relate to the clinical correlation of CD38 expression with poor clinical outcome. The 2H2O labeling technique provides a safe in vivo method to further fractionate the proliferative leukemic compartment as a means to better understand the pathophysiology of B-CLL and possibly provide an important target for therapy.