B-leukemic Cells Research Articles

Identification and characterization of a murine model of BCR‑ABL1+ acute B‑lymphoblastic leukemia with central nervous system metastasis.

Breakpoint cluster region (BCR)-Abelson murine leukemia (ABL)1+ acute B-lymphoblastic leukemia (B-ALL) is a disease associated with a dismal prognosis and a high incidence of central nervous system (CNS) metastasis. However, BCR-ABL1+ B-ALL with CNS infiltration has not been previously characterized, at least to the best of our knowledge. In the present study, a murine model of BCR-ABL1+ B-ALL with CNS metastasis was established using retroviral transduction. The vast majority of BCR-ABL1+ leukemic cells were found to be immature B cells with a variable proportion of pro-B and pre-B populations. The present results indicated that the BCR-ABL1+ B-leukemic cells expressed high levels integrin subunit alpha 6 (Itga6) and L-selectin adhesion molecules, and have an intrinsic ability to disseminate and accumulate in CNS tissues, predominantly in meninges. On the whole, these results provide an approach for addressing the mechanisms of BCR-ABL1+ B-ALL with CNS metastasis and may guide the development of novel therapeutic strategies.

Nanoparticles Engineered with Rituximab and Loaded with Nutlin-3 Show Promising Therapeutic Activity in B-Leukemic Xenografts

Because the nongenotoxic inhibitor of the p53/MDM2 interactions Nutlin-3 has shown promising in vitro therapeutic activity against a variety of p53(wild-type) cancer cells, in this study we evaluated an innovative strategy able to specifically target Nutlin-3 toward CD20(+) malignant cells. The cytotoxic effects of Nutlin-3 encapsulated into poly(lactide-co-glycolide) nanoparticles (NP-Nut) and into rituximab (anti-CD20 antibody)-engineered NP (NP-Rt-Nut) as well as of NPs engineered with rituximab alone (NP-Rt) were initially analyzed in vitro in JVM-2 B-leukemic cells, by assessing both the functional activation of the p53 pathway (by Nutlin-3) and/or the activation of the complement cascade (by rituximab). Moreover, the potential therapeutic efficacy of NP-Nut, NP-Rt, and NP-Rt-Nut were comparatively assessed in vivo in CD20(+) JVM-2 leukemic xenograft SCID mice. Functional in vitro assays showed that NP-Nut and NP-Rt-Nut exhibited a comparable ability to activate the p53 pathway in the p53(wild-type) JVM-2 leukemic cells. On the other hand, NP-Rt and NP-Rt-Nut, but not NP nor NP-Nut, were able to promote activation of the complement cascade. Of note, the in vivo intratumoral injection in JVM-2 B-leukemic/xenograft mice showed that NP-Rt-Nut displayed the maximal therapeutic activity promoting a survival rate significantly higher not only with respect to control animals, treated either with vehicle or with empty NP, but also with respect to animals treated with NP-Nut or NP-Rt. Our data show for the first time the potential antileukemic activity of rituximab-engineered Nutlin-3-loaded NPs in xenograft SCID mice.

Improved Therapy of Multiple Myeloma by Combining Milatuzumab (Anti-CD74 Humanized mAb) and Bortezomib: In Vitro and In Vivo Results.

Background: The humanized anti-CD74 monoclonal antibody, milatuzumab (hLL1, or IMMU-115; Immunomedics, Inc, Morris Plains, NJ), is in clinical evaluation for therapy of multiple myeloma (MM) after preclinical evidence of activity in this tumor type (Stein et al, Blood 2004; 104:3705). Here we examine the ability of milatuzumab to increase the efficacy of drugs in MM cell lines.Methods: MTT cytotoxicity assays were performed on a panel of MM cell lines, including CAG, KMS11, KMS12-PE, and MC/CAR, to examine the effects of bortezomib, doxorubicin (dox), and dexamethasone (dex) alone and combined with milatuzumab or milatuzumab + crosslinking 2nd Ab (goat anti-human IgG, GAH). In vivo studies used a CAG-SCID mouse model of disseminated disease.Results: Without drugs, crosslinked milatuzumab, but not milatuzumab alone, yielded significant anti-proliferative effects on the four MM cell lines. In combination studies, crosslinked milatuzumab produced significant reductions in the IC50 values of the anti-MM drugs. For example, in CAG, milatuzumab+GAH decreased the IC50 values 58%, 78%, and 98% for bortezomib, dox, and dex, respectively (P=0.0034, 0.0073, and 0.078, respectively). In vivo, milatuzumab at 100 μg/injection, 2x weekly for 4 weeks, starting 1 day after injection of CAG cells, more than doubled the median survival time (MST) from 42 days in untreated CAG-bearing SCID mice to 103 days. Combination therapy with milatuzumab and bortezomib or dox was compared to milatuzumab alone, with treatments initiated 5 days after injection of CAG cells. Bortezomib alone (1.0 mg/kg) increased MST from 33 to 44 days (P=0.0021 vs. untreated). Treatment with milatuzumab alone (100 μg/mouse) increased the MST to 73 days (P<0.0001 vs. untreated). When bortezomib and milatuzumab treatments were combined, the MST increased to 93 days (P=0.0441 vs. milatuzumab and P=0.0065 vs. bortezomib). Thus, the combination of milatuzumab and bortezomib increased survival significantly compared to either single treatment. Given alone, dox yielded little or no effect on survival compared with untreated animals, and there was no significant difference between milatuzumab monotherapy and milatuzumab plus doxorubicin in this model. In contrast, a milatuzumabdox immunoconjugate was found to be a highly effective therapeutic agent, with all mice achieving long-term survival. The inhibition of the NF-κB survival pathway of B-leukemic cells by milatuzumab supports its complementary effects when combined with drugs having different mechanisms of action, such as bortezomib.Conclusions: The therapeutic efficacies of bortezomib, dox, and dex are enhanced in vitro in MM cell lines when given in combination with milatuzumab. In vivo, milatuzumab alone or especially in combination with bortezomib is highly effective in MM. (Supported in part by USPHS grant P01CA103985 from the NCI, and grants from the Thomas and Agnes Carvel Foundation and the Walter and Louise Sutcliffe Foundation.)

A Critical Role of Blockade of Cell Differentiation in BCR-ABL-Induced B-Cell Acute Lymphoblastic Leukemia (B-ALL): Basis for Superb Therapeutic Effect on B-ALL in Mice by Promotion of Pro-B Leukemic Cell Differentiation and Treatment with Imatinib.

The BCR-ABL oncogene induces human Philadelphia-positive (Ph+) B-cell acute lymphoblastic leukemia (B-ALL) and chronic myeloid leukemia (CML) that advances to acute phase of CML called blast crisis. In this acute phase, CML patients can develop either B-ALL or acute myeloid leukemia. In B-ALL, differentiation of leukemic cells are blocked at pro-/pre-B stage, and the underlying mechanism is unknown. We hypothesize that this blockade of B-cell differentiation may be important for the development of B-ALL induced by BCR-ABL, and if so, promotion of B-leukemic cell differentiation would create a novel therapeutic strategy for B-ALL. To test this hypothesis, we first compared the percentages of IgM+ B-leukemic cells in BALB/c and C57BL/6 (B6) mice with BCR-ABL-induced B-ALL, because we have previously found that B-ALL develops more quickly in BALB/c mice than in B6 mice (Li et al, J. Exp. Med. 189:1399–1412, 1999). We expressed BCR-ABL in bone marrow (BM) using retroviral transduction and transplantation in these two different strains of inbred mice to induce B-ALL. There were significantly more peripheral blood B220+ B cells in BALB/c B-ALL mice than those in B6 mice, correlating to faster B-ALL in BALB/c mice than in B6 mice. Among these B220+ cells, IgM+ cells were much less in BALB/c mice than in B6 mice. We also compared rearrangement of the B cell antigen receptor (BCR) heavy chains (m chains) between BALB/c and B6 backgrounds using BCR-ABL-expressing pro-B cell lines isolated from the B-ALL mice. Normal m chains rearrangement was found in B6 leukemic cells, but not in BALB/c leukemic cells. These results indicate that more differentiated B-leukemic cells are associated with less aggressive disease. To further demonstrate the role of blockade of B-cell differentiation in B-ALL development, we induced B-leukemic cell differentiation by co-expression of BCR-ABL and intact immunoregulatory tyrosine activation motifs (ITAM) contained in immunoglobulin (Ig)_/Igß complexes in BM cells of B-ALL mice, comparing to expression of BCR-ABL alone. We treated these mice with imatinib (orally, 100 mg/kg, twice a day). The treated mice with B-ALL induced by co-expression of BCR-ABL and ITAM lived three-week longer than those with B-ALL induced by BCR-ABL only, with some mice in long-term remission. Prolonged survival was associated with 50% increased B220+/IgM+ B-leukemic cells in peripheral blood of the mice. Taken together, our results demonstrate that blockade of B-cell differentiation is critical for the development of B-ALL induced by BCR-ABL, and provide a rationale for combination therapy of B-ALL with imatinib and induction of leukemic cell differentiation.

Simultaneous Targeting of SRC and BCR-ABL Kinases by BMS-354825 Cures Ph+ Acute Lymphoblastic Leukemia in Mice.

The ABL kinase inhibitor imatinib mesylate is the preferred treatment for Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) in chronic phase, but is less effective in CML blast crisis or Ph+ B-cell acute lymphoblastic leukemia (B-ALL). We have shown that SRC kinases are required for BCR-ABL-induced B-ALL but not CML (Hu et al., Nat. Genet. , 36:453, 2004), suggesting that SRC kinases may also serve as therapeutic targets in addition to BCR-ABL for B-ALL. To test this idea, we compared the activities of Abl-selective imatinib with BMS-354825, a potent dual inhibitor of SRC and ABL kinases, using the BCR-ABL mutant P210T315I resistant to inhibition by both drugs (Shah et al, Science , 16:399, 2004). We treated P210T315I-expressing pre-B cells with BMS-354825 in vitro. While BMS-354825 did not reduce phosphorylation of P210T315I, it inhibited phosphorylation of SRC kinases at concentrations as low as 25 nM, confirming that BMS-354825 is a potent SRC inhibitor in cells. Moreover, BMS-354825 inhibited proliferation and induced apoptosis of the cells, suggesting that inhibition of SRC kinases alone may suffice in inhibiting the growth of BCR-ABL inhibitor-insensitive leukemic cells. To test whether SRC inhibition was sufficient to exert a therapeutic effect on P210T315I-induced B-ALL, donor bone marrow cells from BALB/c mice were transduced with P210T315I-IRES-GFP retrovirus, followed by transplantation into BALB/c recipients. 8 days after the transplantation, BMS-354825 was given orally twice a day at a dose of 30 mg/kg for 6 days, followed by switching to 10 mg/kg for 3 days and then back to 30mg/kg. Imatinib was given orally twice a day at a dose of 100mg/kg. At day 17 post BMT, the mean peripheral blood (PB) GFP+/B220+ cell count (/ml) was: 1.8x107 for placebo; 0.7x107 for BMS-354825; and 3.0x107 for imatinib. So far (30 days post BMT), 50% (3/6) of placebo-treated and 69% (9/13) of imatinib-treated mice died, whereas all BMS-354825-treated mice are alive. These results suggested that SRC kinases could serve as therapeutic targets for B-ALL although targeting SRC alone may not cure the disease. Similarly, targeting BCR-ABL alone with imatinib could not cure B-ALL. To test dual-target therapy of B-ALL with BMS-354825, we treated mice with B-ALL induced by wild type BCR-ABL. Compared to placebo- and imatinib-treated mice at day 17 post BMT, GFP+/B220+ cells in BMS-354825-treated mice were no longer detectable while PB GFP+/B220+ cells for placebo- and imatinib-treated groups were 3.6x107and 2.4x107/ml, respectively. Although this therapeutic experiment is still ongoing, complete eradication of leukemic cells in BMS-354825-treated mice demonstrates that blockade of both SRC kinases and BCR-ABL cured B-ALL. To understand how SRC kinases function in B-leukemic cells, we looked for SRC downstream signaling molecules using mice deficient for SRC kinases LYN, HCK or FGR. The activation of AKT and STAT5 but not ERK and JNK was down-regulated in leukemic cells lacking LYN, HCK and FGR. The lack of HCK or FGR but not LYN was responsible for their down-regulation. Together, these results indicate that SRC kinases are valuable therapeutic targets for Ph+ B-ALL, and that simultaneous inhibition of SRC and BCR-ABL kinases and their downstream pathways may be beneficial to Ph+ B-ALL patients.

Interferon-gamma increases the expression of glycosylated CD95 in B-leukemic cells: an inducible model to study the role of glycosylation in CD95-signalling and trafficking.

B-lineage acute leukaemia cells are generally resistant to CD95-mediated apoptosis. In this report, the CD95-resistant B-leukaemia lines SEM, RS4;11, and REH were used to investigate the mechanisms of resistance to CD95-signalling. We found that interferon-gamma (IFN-gamma) treatment increased the presence of high molecular weight forms of CD95 in these cells as judged by Western analysis, and treatment of protein extracts with Peptide: N -glycosidase F indicated that the majority of high molecular weight forms were due to N-linked glycosylation. Treatment of whole cells with neuraminidase from Vibrio cholerae substantially reduced the relative molecular mass of CD95 observed after IFN-gamma treatment and partially sensitized the three leukaemia lines to CD95-mediated death. To further characterize the different steps of oligosaccharide processing that may regulate CD95 signalling, the leukaemic cells were treated with IFN-gamma and the glycosidase inhibitors castanospermine, 1-deoxymannojirimycin (DMM), and swainsonine. Treatment with DMM, a mannosidase inhibitor, efficiently reduced the appearance of high molecular weight forms of CD95 after IFN-gamma treatment, and sensitized SEM and REH cells to CD95-mediated death. However, the IFN-gamma-induced increases of CD95 on the cell surface were not altered by treatment with any of the glycosidase inhibitors, suggesting that the generation of complex oligosaccharide structures is not required for trafficking of CD95, but may instead be used as a mechanism of partially blocking CD95 signalling in these cells. In conclusion, IFN-gamma treatment of the B-lineage leukaemia lines provides a novel, inducible system for the further characterization of post-translational modifications involved in modulating sensitivity to CD95-signalling.

Interleukin-6 Inhibits the Proliferation of B-Chronic Lymphocytic Leukemia Cells That Is Induced by Tumor Necrosis Factor-α or -β

Tumor necrosis factor (TNF-alpha) acts as a growth stimulatory factor on leukemic B lymphocytes from many patients with chronic lymphocytic leukemia (CLL). Because TNF induces production of interleukin-6 (IL-6), which has been shown to be a growth factor for myeloma and other transformed B cells, we examined the possibility that IL-6 mediates the growth-stimulatory effect of TNF on B-CLL cells. In fact, we found that IL-6 is an inhibitor of B-CLL growth. The addition of recombinant human IL-6 markedly decreased the TNF-induced B-CLL growth, and this decrease was even greater when soluble IL-6 receptor, known to act as IL-6 agonist, was added with recombinant IL-6. Conversely, neutralizing monoclonal antibodies to IL-6 and to the IL-6 receptor potentiated the growth stimulation of TNF on B-CLL cells, in line with the possibility that IL-6 functions as a negative feedback regulator of an autocrine TNF action on these B-leukemic cells. Evidence is presented that production of IL-6 by monocytes and B cells of CLL patients is low, suggesting that administration of IL-6 may be beneficial in CLL to reduce the eventual growth stimulation by TNF and, possibly, also the deficiency in platelets and Ig production in this disease.

Interleukin-6 inhibits the proliferation of B-chronic lymphocytic leukemia cells that is induced by tumor necrosis factor-alpha or -beta

Tumor necrosis factor (TNF-alpha) acts as a growth stimulatory factor on leukemic B lymphocytes from many patients with chronic lymphocytic leukemia (CLL). Because TNF induces production of interleukin-6 (IL-6), which has been shown to be a growth factor for myeloma and other transformed B cells, we examined the possibility that IL-6 mediates the growth-stimulatory effect of TNF on B-CLL cells. In fact, we found that IL-6 is an inhibitor of B-CLL growth. The addition of recombinant human IL-6 markedly decreased the TNF-induced B-CLL growth, and this decrease was even greater when soluble IL-6 receptor, known to act as IL-6 agonist, was added with recombinant IL-6. Conversely, neutralizing monoclonal antibodies to IL-6 and to the IL-6 receptor potentiated the growth stimulation of TNF on B-CLL cells, in line with the possibility that IL-6 functions as a negative feedback regulator of an autocrine TNF action on these B-leukemic cells. Evidence is presented that production of IL-6 by monocytes and B cells of CLL patients is low, suggesting that administration of IL-6 may be beneficial in CLL to reduce the eventual growth stimulation by TNF and, possibly, also the deficiency in platelets and Ig production in this disease.

Induction of apoptotic cell death in chronic lymphocytic leukemia by 2- chloro-2'-deoxyadenosine and 9-beta-D-arabinosyl-2-fluoroadenine

2-Chloro-2'-deoxyadenosine (CldAdo) and 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A) have shown marked activity in the treatment of indolent lymphoid malignancies. Based on the susceptibility of various lymphocyte populations to apoptosis, we investigated whether CldAdo or F-ara-A would induce this process in lymphocytes from patients with chronic lymphocytic leukemia (CLL). In vitro exposure of leukemic lymphocytes to CldAdo or F-ara-A for 24 to 72 hours elicited features of apoptosis visible by light and electron microscopy. Analysis of DNA integrity showed DNA cleavage into nucleosomal-sized multimers. Using a quantitative assay, drug-induced DNA fragmentation was both time and dose dependent. Inhibition of active macromolecular synthesis did not prevent drug-induced fragmentation; however, both drug-induced and spontaneous DNA fragmentation were prevented by intracellular calcium chelation. In vitro culture with phorbol ester generally decreased drug-induced DNA cleavage. After prolonged incubation, CLL cells exhibited spontaneous cleavage; albeit, at significantly lower rates than drug-treated cells. Heterogeneity was observed for spontaneous and drug-induced DNA fragmentation and was significantly lower in B-leukemic cells obtained from patients with high-risk and refractory disease. We conclude that CldAdo and F-ara-A are potent inducers of apoptotic death in CLL and that this feature correlates with the disease status.

Induction of Apoptotic Cell Death in Chronic Lymphocytic Leukemia by 2-Chloro-2′-deoxyadenosine and 9-β-D-Arabinosyl-2-fluoroadenine

Abstract 2-Chloro-2′-deoxyadenosine (CldAdo) and 9-beta-D-arabinosyl-2- fluoroadenine (F-ara-A) have shown marked activity in the treatment of indolent lymphoid malignancies. Based on the susceptibility of various lymphocyte populations to apoptosis, we investigated whether CldAdo or F-ara-A would induce this process in lymphocytes from patients with chronic lymphocytic leukemia (CLL). In vitro exposure of leukemic lymphocytes to CldAdo or F-ara-A for 24 to 72 hours elicited features of apoptosis visible by light and electron microscopy. Analysis of DNA integrity showed DNA cleavage into nucleosomal-sized multimers. Using a quantitative assay, drug-induced DNA fragmentation was both time and dose dependent. Inhibition of active macromolecular synthesis did not prevent drug-induced fragmentation; however, both drug-induced and spontaneous DNA fragmentation were prevented by intracellular calcium chelation. In vitro culture with phorbol ester generally decreased drug- induced DNA cleavage. After prolonged incubation, CLL cells exhibited spontaneous cleavage; albeit, at significantly lower rates than drug- treated cells. Heterogeneity was observed for spontaneous and drug- induced DNA fragmentation and was significantly lower in B-leukemic cells obtained from patients with high-risk and refractory disease. We conclude that CldAdo and F-ara-A are potent inducers of apoptotic death in CLL and that this feature correlates with the disease status.

No major monoclonal lymphocyte population in the thyroid of patients with Graves' disease: study of gene rearrangement by restriction fragment length polymorphism and polymerase chain reaction.

The present study aimed at determining the mono-, oligo-, or polyclonal nature of intrathyroid lymphocytes at the DNA level in patients with Graves' disease. Two techniques were used to seek monoclonal rearrangement in DNA derived from intrathyroidal lymphocytes obtained from six patients. The first was restriction fragment length polymorphism using two specific probes from the B-chain of T-cell receptor and the other from the heavy chain immunoglobulin gene; the second was polymerase chain reaction using a couple of specific primers from the variable and joining regions of heavy chain immunoglobulins. The results for the patients with Graves' disease were compared with those obtained for circulating T-and B-lymphocytes, granulocytes (negative controls), and T- and B-leukemic cells (positive controls). The results with restriction fragment length polymorphism favored a polyclonal origin for the lymphocytes in all cases, since no rearrangement was visualized. The results with polymerase chain reaction were analogous, and the technique was 10 times more sensitive in the detection of rearrangement.

A new supertypic HLA class II determinant (PL2) differentially expressed with DR7, DRw11(5), DRw13(w6), DRw14(w6), DR3, and DR2 and biochemically localized to DR7 molecules

A monoclonal antibody, PL2, has been produced that reacts with a new supertypic determinant expressed on the peripheral blood B lymphocytes and B-leukemic cells (B-CLL) from all individuals who are HLA-DR7 and some individuals who are HLA-DR5 positive. The genetic linkage of the PL2 determinant to the HLA region was demonstrated by family segregation studies. When cultured Epstein-Barr virus (EBV) transformed B cell lines were examined, PL2 was again found to be expressed on all cell lines homozygous for HLA-DR7 and the DRw11(5) subtype of HLA-DR5 positive cells, while one DRw12(5) cell line was negative, suggesting PL2 may distinguish between these DR5 subtypes. In addition, using the panel of EBV-transformed B-cell lines, PL2 was also found to be weakly expressed on HLA-DRw14(w6), -DRw13(w6), -DR3, and -DR2 positive cells but was completely absent from HLA-DR1 and -DR4 positive cells, and is probably absent also from DRw8- and DRw10-positive cells. From titration analysis and quantitative absorption studies the PL2 determinant was found to be expressed at quantitatively different levels in the following order: DR7> DRw11, DRw14>DRw13>Dr3>DR2. The molecules carrying the PL2 determinant on DR7 cells have been characterized biochemically to be a subpopulation of HLA class II molecules recognized by the DR specific monoclonal antibody, L243. Furthermore, by two-dimensional gel analysis, PL2 immunoprecipitated only two of three beta chains associated with the DR-alpha chain, which are the same two chains that carry the DR7 allodeterminants. Thus, PL2 was found to recognized a new supertypic determinant, which on DR7 cell lines is expressed on a subpopulation of HLA-DR molecules.

TPA-Induced Maturation in Secretory Human B-Leukemic Cells In Vitro: DNA Synthesis, Antigenic Changes, and Immunoglobulin Secretion

The maturation of malignant cells in response to differentiating agents is interesting as a model of normal differentiation. The response of a freshly explanted neoplastic population of phenotypically well-characterized lymphosarcoma cell leukemia blasts was studied after incubation with the differentiating agent TPA (12-0-tetradecanoyl-phorbol-13-acetate). Terminal differentiation was assessed by measuring the immunoglobulin secreted in culture supernatants and the production of intracytoplasmic immunoglobulins. Activation of the cells was studied using fluorescein-labeled monoclonal antibodies to various antigens in a flow cytometer (fluorescence-activated cell sorter) and 3H-thymidine (3H-Tdr) incorporation was evaluated to measure DNA synthesis in cells grown in complete medium and TPA-supplemented medium. The events induced by TPA were characteristic of B cell maturation and included morphological changes to plasmacytoid cells, reduction in surface immunoglobulins (sIgM, sIgD, and K), enhancement of cytoplasmic immunoglobulin, and amplification of immunoglobulin secretion. Surface antigen changes were accompanied by increased 3H-Tdr incorporation. Cell proliferation and differentiation appeared to be coupled and both were amplified by TPA treatment. These observations indicate that TPA can promote maturation of malignant secretory B cells to a terminal differentiation stage. The significance of these findings to normal B cell differentiation and their potential clinical utility is discussed.

Tpa-induced maturation in secretory human B-leukemic cells in vitro: DNA synthesis, antigenic changes, and immunoglobulin secretion

The maturation of malignant cells in response to differentiating agents is interesting as a model of normal differentiation. The response of a freshly explanted neoplastic population of phenotypically well- characterized lymphosarcoma cell leukemia blasts was studied after incubation with the differentiating agent TPA (12–0-tetradecanoyl- phorbol-13-acetate). Terminal differentiation was assessed by measuring the immunoglobulin secreted in culture supernatants and the production of intracytoplasmic immunoglobulins. Activation of the cells was studied using fluorescein-labeled monoclonal antibodies to various antigens in a flow cytometer (fluorescence-activated cell sorter) and 3H-thymidine (3H-Tdr) incorporation was evaluated to measure DNA synthesis in cells grown in complete medium and TPA-supplemented medium. The events induced by TPA were characteristic of B cell maturation and included morphological changes to plasmacytoid cells, reduction in surface immunoglobulins (sIgM, sIgD, and K), enhancement of cytoplasmic immunoglobulin, and amplification of immunoglobulin secretion. Surface antigen changes were accompanied by increased 3H-Tdr incorporation. Cell proliferation and differentiation appeared to be coupled and both were amplified by TPA treatment. These observations indicate that TPA can promote maturation of malignant secretory B cells to a terminal differentiation stage. The significance of these findings to normal B cell differentiation and their potential clinical utility is discussed.