A tumor-related lymphoid progenitor population supports hierarchical tumor organization in canine B-cell lymphoma.

BACKGROUND: Tumors have heterogeneous properties, which could be explained by the existence of hierarchically and biologically distinct tumor cells such as tumor-initiating cells (TICs). This model is clinically important, as TICs are promising targets for cancer therapies. However, TICs in spontaneous B-cell lymphoma have not been conclusively identified. HYPOTHESIS/OBJECTIVES: Tumor cells with a progenitor phenotype exist in B-cell lymphoma, reflecting a hierarchical organization. ANIMALS: Twenty-eight client-owned dogs with previously untreated B-cell lymphoma and 6 healthy dogs. METHODS: This was a prospective study. Flow cytometry was used to identify lymphoid progenitor cells (LPCs) that coexpressed hematopoietic progenitor antigens CD34, CD117, and CD133, with lymphoid differentiation markers CD21 and/or CD22 in B-cell lymphoma. The polymerase chain reaction for antigen receptor rearrangements was used to analyze clonality and relatedness of tumor populations. A xenograft model with NOD/SCID/IL-2Rγ(-/-) mice was adapted to expand and serially transplant primary canine B-cell lymphoma. RESULTS: LPCs were expanded in lymph nodes from 28 dogs with B-cell lymphoma compared with 6 healthy dogs (P= .0022). LPCs contained a clonal antigen receptor gene rearrangement identical to that of the bulk of tumor cells. Canine B-cell lymphoma xenografts in recipient mice that maintained LPCs in the tumors were recurrently observed. CONCLUSIONS AND CLINICAL IMPORTANCE: These results suggest the presence of a hierarchy of tumor cells in B-cell lymphoma as has been demonstrated in other cancers. These findings have the potential to impact not only the understanding of lymphoma pathogenesis but also the development of lymphoma therapies by providing novel targets for therapy.

Suppression of apoptosis induced by growth factor withdrawal by an oncogenic form of c-Cbl.

The v-Cbl oncogene induces myeloid and B-cell leukemia; however, the mechanism by which transformation occurs is not understood. An oncogenic form of c-Cbl (Cbl-DeltaY371) was expressed in the interleukin-3 (IL-3)-dependent cell line 32Dcl3 to determine whether it was able to induce growth factor-independent proliferation. We were unable to isolate clones of transfected 32Dcl3 cells expressing Cbl-DeltaY371 that proliferated in the absence of IL-3. In contrast, 32Dcl3/Cbl-DeltaY371 cells did not undergo apoptosis like parental 32Dcl3 cells when cultured in the absence of IL-3. Both 32Dcl3 and 32D/CblDeltaY371 cells arrested in G(1) when cultured in the absence of IL-3. Approximately 18% of the 32Dcl3 cells cultured in the absence of IL-3 for 24 h were present in a sub-G(1) fraction, while only 4% of the 32D/Cbl-DeltaY371 and 2% of the 32D/Bcl-2 cells were found in a sub-G(1) fraction. There was no difference in the pattern of tyrosine-phosphorylated proteins observed following stimulation of either cell type with IL-3. The phosphorylation of JAK2, STAT5, and endogenous c-Cbl was identical in both cell types. No differences were detected in the activation of Akt, ERK1, or ERK2 in unstimulated or IL-3-stimulated 32D/Cbl-DeltaY371 cells compared with parental 32Dcl3 cells. Likewise, there was no difference in the pattern of phosphorylation of JAK2, STAT5, ERK1, ERK2, or Akt when 32Dcl3 and 32D/CblDY371 cells were withdrawn from medium containing IL-3. The protein levels of various Bcl-2 family members were examined in cells grown in the absence or presence of IL-3. We observed a consistent increased amount of Bcl-2 protein in five different clones of 32D/Cbl-DeltaY317 cells. These data suggest that the Cbl-DeltaY371 mutant may suppress apoptosis by a mechanism that involves the overexpression of Bcl-2. Consistent with this result, activation of caspase-3 was suppressed in 32D/Cbl-DeltaY371 cells cultured in the absence of IL-3 compared with 32Dcl3 cells cultured under the same conditions.

Peroxidase activity in murine and human hematopoietic progenitor cells: potential relevance to benzene-induced toxicity.

Peroxidases may be important in the mechanism of toxicity of a number of compounds including benzene, a chemical that has been associated with bone marrow toxicity and leukemia after chronic exposure. The major peroxidase in bone marrow is myeloperoxidase (MPO), which has been previously thought to be expressed at the promyelocytic stage of differentiation. Hematopoietic progenitor cells are important potential cellular targets of bone marrow toxins and leukemogens. We therefore examined peroxidase activity in both murine and human progenitor cells. Murine progenitor populations were purified as lineage-negative cells (> 99% enriched) and human progenitor populations were purified as CD34+ cells (> 95% enriched). Using conventional biochemical assays for peroxidase activity, murine and human progenitor cells were found to have 30% and 11% of the peroxidase activity of murine and human unpurified marrow, respectively. Peroxidase activity was confirmed in purified murine and human progenitor populations by flow cytometry using a 2,7-dichlorofluorescein assay, adapted to measure peroxidase activity. In addition, two-color flow cytometry of murine whole marrow using phycoerythrin-conjugated antibodies to lineage markers confirmed the peroxidase activity of the murine progenitor cell population. A reverse transcription-polymerase chain reaction assay was developed for MPO mRNA, which was detected in murine progenitor cells. These data show that MPO mRNA is expressed in murine progenitor cells and that both murine and human progenitor cells have marked peroxidase activity. These data may have relevance for studies of hematopoietic cell differentiation and for the examination of mechanisms underlying cell-specific toxicity in bone marrow.