Value of flow cytometry for MRD-based relapse prediction in B-cell precursor ALL in a multicenter setting.

PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR5y) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p < 0.0001), 29 (HzR 2.7, p < 0.0001), and 79 (HzR 3.5, p < 0.0001) associated with hazard of relapse adjusted for age, cytogenetics, and WBC. The early (day 15) response associated with CIR5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10-2 versus 5.2 × 10-3, p < 0.0001). Undetectable FCM- and/or PCR-MRD on day 29 identified patients with a very good outcome (CIR5y = 3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD > 10-4 associated with a CIR5y = 22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.

Modeling chronic myeloid leukemia in immunodeficient mice reveals expansion of aberrant mast cells and accumulation of pre-B cells.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that, if not treated, will progress into blast crisis (BC) of either myeloid or B lymphoid phenotype. The BCR-ABL1 fusion gene, encoding a constitutively active tyrosine kinase, is thought to be sufficient to cause chronic phase (CP) CML, whereas additional genetic lesions are needed for progression into CML BC. To generate a humanized CML model, we retrovirally expressed BCR-ABL1 in the cord blood CD34(+) cells and transplanted these into NOD-SCID (non-obese diabetic/severe-combined immunodeficient) interleukin-2-receptor γ-deficient mice. In primary mice, BCR-ABL1 expression induced an inflammatory-like state in the bone marrow and spleen, and mast cells were the only myeloid lineage specifically expanded by BCR-ABL1. Upon secondary transplantation, the pronounced inflammatory phenotype was lost and mainly human mast cells and macrophages were found in the bone marrow. Moreover, a striking block at the pre-B-cell stage was observed in primary mice, resulting in an accumulation of pre-B cells. A similar block in B-cell differentiation could be confirmed in primary cells from CML patients. Hence, this humanized mouse model of CML reveals previously unexplored features of CP CML and should be useful for further studies to understand the disease pathogenesis of CML.

SOCS2 is dispensable for BCR/ABL1-induced chronic myeloid leukemia-like disease and for normal hematopoietic stem cell function.

Suppressor of cytokine signaling 2 (SOCS2) is known as a feedback inhibitor of cytokine signaling and is highly expressed in primary bone marrow (BM) cells from patients with chronic myeloid leukemia (CML). However, it has not been established whether SOCS2 is involved in CML, caused by the BCR/ABL1 fusion gene, or important for normal hematopoietic stem cell (HSC) function. In this study, we demonstrate that although Socs2 was found to be preferentially expressed in long-term HSCs, Socs2-deficient HSCs were indistinguishable from wild-type HSCs when challenged in competitive BM transplantation experiments. Furthermore, by using a retroviral BCR/ABL1-induced mouse model of CML, we demonstrate that SOCS2 is dispensable for the induction and propagation of the disease, suggesting that the SOCS2-mediated feedback regulation of the JAK/STAT pathway is deficient in BCR/ABL1-induced CML.

Origins of cheating and loss of symbiosis in wild Bradyrhizobium.

Rhizobial bacteria nodulate legume roots and fix nitrogen in exchange for photosynthates. These symbionts are infectiously acquired from the environment and in such cases selection models predict evolutionary spread of uncooperative mutants. Uncooperative rhizobia - including nonfixing and non-nodulating strains - appear common in agriculture, yet their population biology and origins remain unknown in natural soils. Here, a phylogenetically broad sample of 62 wild-collected rhizobial isolates was experimentally inoculated onto Lotus strigosus to assess their nodulation ability and effects on host growth. A cheater strain was discovered that proliferated in host tissue while offering no benefit; its fitness was superior to that of beneficial strains. Phylogenetic reconstruction of Bradyrhizobium rDNA and transmissible symbiosis-island loci suggest that the cheater evolved via symbiotic gene transfer. Many strains were also identified that failed to nodulate L. strigosus, and it appears that nodulation ability on this host has been recurrently lost in the symbiont population. This is the first study to reveal the adaptive nature of rhizobial cheating and to trace the evolutionary origins of uncooperative rhizobial mutants.

Mitochondrial DNA variation along an altitudinal gradient in the greater white-toothed shrew, Crocidura russula.

The distribution of mitochondrial control region-sequence polymorphism was investigated in 15 populations of Crocidura russula along an altitudinal gradient in western Switzerland. High-altitude populations are smaller, sparser and appear to undergo frequent bottlenecks. Accordingly, they showed a loss of rare haplotypes, but unexpectedly, were less differentiated than lowland populations. Furthermore, the major haplotypes segregated significantly with altitude. The results were inconsistent with a simple model of drift and dispersal. They suggested instead a role for historical patterns of colonization, or, alternatively, present-day selective forces acting on one of the mitochondrial genes involved in metabolic pathways.

Influence of CD4 or CD8 deficiency on collagen-induced arthritis.

The role of T cells in the mouse collagen-induced arthritis (CIA) model for rheumatoid arthritis is not clarified, and different results have been reported concerning the role of CD4 and CD8 T cells. To address this issue, we have investigated B10.Q mice deficient for CD4 or CD8. The mice lacking CD4 were found to be less susceptible to disease, but not completely resistant, whereas the CD8 deficiency had no significant impact on the disease. No difference in the development of late occurring relapses was noted. Interestingly, the CD4-deficient mice had a severely reduced response to the glycosylated form of the immunodominant type II collagen (CII) 256-270 peptide whereas the response to the non-glycosylated peptide was not significantly different. Furthermore, CD4-deficient mice had lower antibody responses to CII, explaining the lower disease susceptibility. In comparison with previously reported results, it is apparent that the lack of CD4 molecules has a different impact on CIA if present on different genetic backgrounds, findings that could possibly be related to the occurrence of different disease pathways of CIA in different mouse strains.

Characterization of the molecular mechanisms for p53-mediated differentiation.

The p53 tumor suppressor protein can induce both apoptosis and cell cycle arrest. Moreover, we and others have shown previously that p53 is a potent mediator of differentiation. For example, expression of ptsp53, a temperature-inducible form of p53, induces differentiation of leukemic monoblastic U-937 cells. The functions of p53 have for long been believed to be dependent on the transactivating capacity of p53. However, recent data show that both p53-induced cell cycle arrest and apoptosis can be induced independently of p53-mediated transcriptional activation, indicating alternative pathways for p53-induced apoptosis and cell cycle arrest. The bcl-2 proto-oncogene contributes to the development of certain malignancies, probably by inhibition of apoptosis. Interestingly, Bcl-2 has been shown to inhibit p53-mediated apoptosis as well as p53-mediated transcriptional activation. Asking whether Bcl-2 would interfere with the p53-mediated differentiation of U-937 cells, we stably transfected bcl-2 to U-937 cells inducibly expressing p53. Although the established Bcl-2-expressing clones were resistant to p53-mediated apoptosis, we did not observe any interference of Bcl-2 with the p53-mediated differentiation, suggesting separable pathways for p53 in mediating apoptosis and differentiation of U-937 cells. Neither did expression of Bcl-2 interfere with p53-induced expression of endogenous p21, suggesting that p53-induced differentiation might be dependent on the transcriptional activity of p53. To further investigate whether the p53-mediated differentiation of U-937 cells depends on the transcriptional activity of p53, we overexpressed transactivation-deficient p53, a transcriptionally inactive p53 mutant in these cells. However, in contrast to the effects of wild-type p53, expression of trans-activation-deficient p53 did neither induce signs of apoptosis nor of differentiation in U-937 cells. Our results indicate that the transcriptional activity of p53 is essential both for p53-mediated apoptosis and differentiation of U-937 cells.

p53-mediated differentiation of the erythroleukemia cell line K562.

The tumor suppressor gene p53 can mediate both apoptosis and cell cycle arrest. In addition, p53 also influences differentiation. To further characterize the differentiation inducing properties of p53, we overexpressed a temperature-inducible p53 mutant (ptsp53Val135) in the erythroleukemia cell line K562. The results show that wild-type p53 and hemin synergistically induce erythroid differentiation of K562 cells, indicating that p53 plays a role in the molecular regulation of differentiation. However, wild-type p53 did not affect phorbol 12-myristate 13-acetate-dependent appearance of the megakaryocyte-related cell surface antigens CD9 and CD61, suggesting that p53 does not generally affect phenotypic modulation. The cyclin-dependent kinase inhibitor p21, a transcriptional target of p53, halts the cell cycle in G1 and has also been implicated in the regulation of differentiation and apoptosis. However, transiently overexpressed p21 did neither induce differentiation nor affect the cell cycle distribution or viability of K562 cells, suggesting that targets downstream of p53 other than p21 are critical for the p53-mediated differentiation response.

c-Myc antagonizes the effect of p53 on apoptosis and p21WAF1 transactivation in K562 leukemia cells.

c-myc protooncogene positively regulates cell proliferation and overexpression of c-myc is found in many solid tumors and leukemias. In the present study we used the K562 human myeloid leukemia cell line as a model to study the functional interaction between c-Myc and p53. Using two different methods, we generated K562 transfectant cell lines with conditional expression of either c-Myc or p53. The cells expressed the p53Vall35 mutant, which adopts a wild-type conformation at 32 degrees C, while c-Myc induction was achieved with a zinc-inducible expression vector. We found that p53 in wild-type conformation induces growth arrest and apoptosis of K562. Expression of c-Myc significantly attenuated apoptosis and impaired the transcriptional activity of p53 on p21WAF1, Bax and cytomegalovirus promoters. The impairment of p21WAF1 transactivation by c-Myc was confirmed by transfection of a c-Myc-estrogen receptor fusion protein and by induction of c-myc by zinc in transfected cells. Also, p53-mediated up-regulation of p21WAF1 mRNA protein were significantly reduced by c-Myc, while Bax levels were unaffected. Consistently, c-Myc increased cyclin-dependent kinase 2 activity in K562 cells expressing p53 in wild-type conformation. These results suggest that c-Myc overexpression may antagonize the pro-apoptotic function of p53, thus providing a molecular mechanism for the frequently observed deregulation of c-myc in human cancer.

Induction of apoptosis in U937 human leukemia cells by suberoylanilide hydroxamic acid (SAHA) proceeds through pathways that are regulated by Bcl-2/Bcl-XL, c-Jun, and p21CIP1, but independent of p53.

Determinants of differentiation and apoptosis in myelomonocytic leukemia cells (U937) exposed to the novel hybrid polar compound SAHA (suberoylanilide hydroxamic acid) have been examined. In contrast to hexamethylenbisacetamide (HMBA), SAHA-related maturation was limited and accompanied by marked cytoxicity. SAHA-mediated apoptosis occurred within the G0G1 and S phase populations, and was associated with decreased mitochondrial membrane potential, caspase-3 activation, PARP degradation, hypophosphorylation/cleavage of pRB, and down-regulation of c-Myc, c-Myb, and B-Myb. Enforced expression of Bcl-2 or Bcl-XL inhibited SAHA-induced apoptosis, but only modestly potentiated differentiation. While SAHA induced the cyclin-dependent kinase inhibitor p21CIP1, antisense ablation of this CDKI increased, rather than decreased, SAHA-related lethality. In contrast, conditional expression of wild-type p53 failed to modify SAHA actions, but markedly potentiated HMBA-induced apoptosis. Finally, SAHA modestly increased expression/activation of the stress-activated protein kinase (SAPK/JNK); moreover, SAHA-related lethality was partially attenuated by a dominant-negative c-Jun mutant protein (TAM67). SAHA did not stimulate mitogen-activated protein kinase (MAPK), nor was lethality diminished by the specific MEK/MAPK inhibitor PD98059. These findings indicate that SAHA potently induces apoptosis in human leukemia cells via a pathway that is p53-independent but at least partially regulated by Bcl-2/Bcl-XL, p21CIP1, and the c-Jun/AP-1 signaling cascade.

Involvement of the retinoblastoma protein in monocytic and neutrophilic lineage commitment of human bone marrow progenitor cells.

The retinoblastoma gene product (pRb) is involved in both cell cycle regulation and cell differentiation. pRb may have dual functions during cell differentiation: partly by promoting a cell cycle brake at G(1) and also by interacting with tissue-specific transcription factors. We recently showed that pRb mediates differentiation of leukemic cell lines involving mechanisms other than the induction of G(1) arrest. In the present study, we investigated the role of pRb in differentiation of human bone marrow progenitor cells. Human bone marrow cells were cultured in a colony-forming unit-granulocyte-macrophage (CFU-GM) assay. The addition of antisense RB oligonucleotides (alpha-RB), but not the addition of sense orientated oligonucleotides (SO) or scrambled oligonucleotides (SCR), reduced the number of colonies staining for nonspecific esterase without affecting the clonogenic growth. Monocytic differentiation of CD34(+) cells supported by FLT3-ligand and interleukin-3 (IL-3) was correlated to high levels of hypophosphorylated pRb, whereas neutrophilic differentiation, supported by granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF), was correlated to low levels. The addition of alpha-RB to liquid cultures of CD34(+) cells, supported with FLT3-ligand and IL-3, inhibited monocytic differentiation. This was judged by morphology, the expression of CD14, and staining for esterase. Moreover, the inhibition of monocytic differentiation of CD34(+) cells mediated by alpha-RB, which is capable of reducing pRb expression, was counterbalanced by an enhanced neutrophilic differentiation response, as judged by morphology and the expression of lactoferrin. CD34(+) cells incubated with oligo buffer, alpha-RB, SO, or SCR showed similar growth rates. Taken together, these data suggest that pRb plays a critical role in the monocytic and neutrophilic lineage commitment of human bone marrow progenitors, probably by mechanisms that are not strictly related to control of cell cycle progression.

Abnormal cell cycle regulation in malignancy.

The cell cycle consists of an initial growth phase (G1), DNA replication (S), a gap phase (G2), and mitosis (M), after which the cell may differentiate or enter the resting state (G0). The cycle is driven by a number of positive and negative regulatory phosphorylation and dephosphorylation events, involving protein kinases, protein phosphatases, cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors, that ultimately impinge on the activity of transcription factors. Unreplicated or damaged DNA blocks the progression of the cell cycle at checkpoints, including a late G1 checkpoint regulated by the dephosphorylated retinoblastoma protein and a late G2 checkpoint regulated by the phosphorylation of cyclin-dependent kinase 1 complexed with cyclin B. Many cell cycle regulator genes may be considered proto-oncogenes or tumor suppressor genes, and point mutations, amplifications, deletions, or rearrangements involving their loci, particularly those in the "RB pathway," are associated with various tumors. A number of molecular techniques may be used to detect genomic alterations or posttranscriptional modifications, but immunohistochemistry remains the most common method to determine expression levels of a regulatory protein. Multivariate analysis of the usefulness in prognosis has been applied most often for the general proliferation antigen Ki-67.

p53-dependent and -independent differentiation of leukemic U-937 cells: relationship to cell cycle control.

Observations based on overexpression of the suppressor gene p53 or interference with endogenous p53 support a role for p53 in mediating not only growth inhibition and apoptosis but also differentiation. The aim of this study was to characterize the mechanisms of p53-dependent differentiation in the monoblastic leukemia cell line U-937. These cells were transfected with a mutant of the p53 gene expressing wild-type p53 at a permissive temperature. The results showed that wild-type p53 and interferon (IFN)-gamma were able to work synergistically to promote differentiation. This cooperative response was not associated with early G1 arrest of the cell cycle, indicating that p53 can mediate differentiation by mechanisms other than those used for mediating G1 arrest. The differentiation response to transfected p53 with or without INF-gamma was inhibited by cyclic adenosine monophosphate (cAMP)-inducing agents (dibutyryl cyclic adenosine 3':5'-monophosphate, forskolin, and 3-isobutyl-1-methylxanthine) in a dose-dependent manner. In contrast, the differentiation response of p53-negative U-937 cells to 1,25-dihydroxychole-calciferol or all-trans retinoic acid was enhanced by cAMP-inducing agents at optimal concentrations and inhibited at higher concentrations. In addition, 1,25-dihydroxycholecalciferol-mediated differentiation could be achieved in cells arrested in G1 by concomitant incubation with cAMP-inducing agents, indicating that differentiation can occur in the absence of proliferation. In conclusion, the results of this study indicate that p53-dependent and -independent differentiation can occur independently of cell cycle regulation.

The tumor suppressor gene p53 can mediate transforming growth [corrected] factor beta1-induced differentiation of leukemic cells independently of activation of the retinoblastoma protein.

Although the involvement of the tumor suppressor gene p53 in normal hematopoiesis is uncertain, it can give rise to differentiation signals in leukemic cells. It is not clear, however, whether differentiation merely is a consequence of the ability of p53 to arrest cell proliferation or whether hitherto unknown molecular mechanisms are responsible for the p53-mediated differentiation. To further explore the role of p53 in leukemic cell differentiation, we investigated whether transforming growth factor beta1 (TGF-beta1), a cytokine involved in cell cycle control at several levels, can cooperate with wild-type p53 to induce differentiation of monoblastic U-937 and erythroleukemic K562 cells. Indeed, wild-type p53-expressing cells were found to be more sensitive to TGF-beta1-induced differentiation than control cells, lending support to the idea that p53 is of importance for differentiation induction of leukemic cells. In addition, it is shown that TGF-beta1 can suppress p53-mediated cell death, thus reinforcing the differentiation response. The cyclin-dependent kinase inhibitor p21 and the retinoblastoma protein (pRb) are downstream effectors of p53-mediated growth arrest. Therefore, the roles for these molecules in p53-mediated differentiation were examined. The p53-dependent signals of differentiation were associated with induction of p21 in both cell lines investigated. However, activation of pRb by induced hypophosphorylation and concomitant decreased growth rate on p53-mediated differentiation was observed only in U-937 cells expressing an inducible, temperature-sensitive form of p53 but not in K562 cells constitutively expressing p53. Thus, our data suggest a role for p53 in the regulation of differentiation in leukemic cells that can be independent of its ability to activate pRb and arrest cell proliferation.

Altered expression of the retinoblastoma tumor-suppressor gene in leukemic cell lines inhibits induction of differentiation but not G1-accumulation.

The retinoblastoma tumor-suppressor gene, RB, has been implicated in tumor suppression, in regulation of the cell cycle, and in mediating cell differentiation. RB is necessary for hematopoiesis in mice, and aberrant RB-expression is associated with the progress and prognosis of leukemia. We have used antisense oligonucleotides, established clones stably expressing an antisense RB construct, and also established clones over expressing the retinoblastoma protein (pRb) to study the role of RB expression in monocytic differentiation induced by all-trans retinoic acid (ATRA) or 1-alpha-25-dihyroxycholecalciferol (Vit D3) in the monoblastic cell line U-937 and erythroid differentiation induced by transforming growth factor beta1 (TGFbeta1) and hemin in the erythroleukemic cell line K562. A reduction in pRb production in antisense RB-transfected U-937 clones was shown. Antisense oligonucleotides as well as expression of the antisense RB construct suppressed differentiation responses to ATRA or Vit D3, as judged by the capability to reduce nitro blue tetrazolium, by the appearance of monocyte-related cell surface antigens and by morphologic criteria. K562 cells showed decreased differentiation response to TGFbeta1, but not to hemin, when incubated with antisense oligonucleotides. U-937 antisense RB-transfected cells were also suppressed in their ability to upregulate levels of hypophosphorylated pRb when induced to differentiate. Although U-937 cells incubated with antisense oligonucleotides and clones expressing the antisense RB construct were hampered in their ability to differentiate on incubation with ATRA or Vit D3, the induced G0/G1-accumulation was similar to differentiating control cells treated with ATRA or Vit D3. Intriguingly, U-937 clones overexpressing RB were also inhibited in their differentiation response to ATRA or Vit D3 but not inhibited in their ability to respond with G0/G1 accumulation when induced with these substances. The results indicate that pRb plays a role in induced differentiation of U-937 cells as well as K562 cells involving mechanisms that, at least partially, are distinct from those inducing G1 accumulation.

Cell differentiation in acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by a differentiation block leading to accumulation of immature cells. Chromosomal translocations in AML affect transcription factors that are involved in regulation of myeloid differentiation. Aberrant expression of these factors interferes with differentiation events and has a role in the pathogenesis of AML through superactivation or (dominant negative) repression of genes regulating proliferation and differentiation or by interference with assembly of the transcription complex for these genes. The maturation arrest can be reversed by certain agents as judged by results from investigations of myeloid leukemic cell lines and from treatment of acute promyelocytic leukemia (APL) patients with all-trans retinoic acid. Inactivation of the p53 and retinoblastoma (Rb) tumor suppressor genes is also associated with the pathogenesis of leukemia through effects on the cell cycle, and manipulation of these genes can affect differentiation of AML cells. With differentiation therapy, when successful as in APL, the leukemic cell mass is reduced to allow restoration of normal hematopoiesis and clinical remission, but the disease is not cured. However, initial reduction of the cell mass by maturation can increase the probability for cure with chemotherapy. Overexpression of suppressor genes may increase the probability for differentiation. Most probably, particular molecular defects of subgroups of AML have to be explored to find optimal strategies for treatment including both blocking the cell cycle, promoting terminal differentiation, and inducing apoptosis as well as strengthening the immune response.

Expression of the p53 tumor suppressor gene induces differentiation and promotes induction of differentiation by 1,25-dihydroxycholecalciferol in leukemic U-937 cells.

Leukemic U-937 cells, which lack normal p53, were stably transfected with a temperature-sensitive mutant of p53 to investigate the consequences for growth and differentiation. On induction of wild-type p53 activity at the permissive temperature, some of these cells underwent maturation as judged by the capacity for oxidative burst and the appearance of monocyte related cell surface molecules. Moreover, wild-type p53-expressing cells were more sensitive than p53-negative control cells to induction of differentiation by 1,25-dihydroxycholecalciferol; a twofold to fourfold increase of the fraction of cells showing signs of terminal maturation was observed when wild-type p53-expressing cells were incubated with 1,25-dihydroxycholecalciferol at concentrations that only slightly affected control cells. Whereas wild-type p53 activity per se induced maturation of certain cells, other underwent cell death judging from the reduced capability to exclude trypan blue and the appearance of fragmented DNA in flow cytometric analysis. The p53-induced cell death could be inhibited by incubation with 1,25-dihydroxy-cholecalciferol, but not all-trans retinoic acid. Thus, 1,25-dihydroxycholecalciferol, seemed to increase the survival of wild-type p53-expressing cells and to cooperate with wild-type p53 to induce differentiation. The data imply that p53-mediated maturation in U-937 cells depends on optimal regulation of signals for differentiation, survival and proliferation, and suggest a role for p53 in the differentiation induction of leukemic cells.