Hypercalcemia in childhood acute lymphoblastic leukemia: frequent implication of parathyroid hormone-related peptide and E2A-HLF from translocation 17;19.

Hypercalcemia is relatively rare but clinically important complication in childhood leukemic patients. To clarify the clinical characteristics, mechanisms of hypercalcemia, response to management for hypercalcemia, incidence of t(17;19) and final outcome of childhood acute lymphoblastic leukemia (ALL) accompanied by hypercalcemia, clinical data of 22 cases of childhood ALL accompanied by hypercalcemia (>12 mg/dl) reported in Japan from 1990 to 2005 were retrospectively analyzed. Eleven patients were 10 years and older. Twenty patients had low white blood cell count (<20 x 10(9)/l), 15 showed hemoglobin> or =8 g/dl and 14 showed platelet count > or =100 x 10(9)/l. Parathyroid hormone-related peptide (PTHrP)-mediated hypercalcemia was confirmed in 11 of the 16 patients in whom elevated-serum level or positive immunohistochemistry of PTHrP was observed. Hypercalcemia and accompanying renal insufficiency resolved quickly, particularly in patients treated with bisphosphonate. t(17;19) or add(19)(p13) was detected in five patients among 17 patients in whom karyotypic data were available, and the presence of E2A-HLF was confirmed in these five patients. All five patients with t(17;19)-ALL relapsed very early. Excluding the t(17;19)-ALL patients, the final outcome of ALL accompanied by hypercalcemia was similar to that of all childhood ALL patients, indicating that the development of hypercalcemia itself is not a poor prognostic factor.

The E2A-HLF oncoprotein activates Groucho-related genes and suppresses Runx1.

The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) chromosomal translocation in leukemic pro-B cells, encodes a chimeric transcription factor consisting of the transactivation domain of E2A linked to the bZIP DNA-binding and protein dimerization domain of hepatic leukemia factor (HLF). This oncoprotein blocks apoptosis induced by growth factor deprivation or irradiation, but the mechanism for this effect remains unclear. We therefore performed representational difference analysis (RDA) to identify downstream genetic targets of E2A-HLF, using a murine FL5.12 pro-B cell line that had been stably transfected with E2A-HLF cDNA under the control of a zinc-regulated metallothionein promoter. Two RDA clones, designated RDA1 and RDA3, were differentially upregulated in E2A-HLF-positive cells after zinc induction. The corresponding cDNAs encoded two WD40 repeat-containing proteins, Grg2 and Grg6. Both are related to the Drosophila protein Groucho, a transcriptional corepressor that lacks DNA-binding activity on its own but can act in concert with other proteins to regulate embryologic development of the fly. Expression of both Grg2 and Grg6 was upregulated 10- to 50-fold by E2A-HLF. Immunoblot analysis detected increased amounts of two additional Groucho-related proteins, Grg1 and Grg4, in cells expressing E2A-HLF. A mutant E2A-HLF protein with a disabled DNA-binding region also mediated pro-B cell survival and activated Groucho-related genes. Among the transcription factors known to interact with Groucho-related protein, only RUNX1 was appreciably downregulated by E2A-HLF. Our results identify a highly conserved family of transcriptional corepressors that are activated by E2A-HLF, and they suggest that downregulation of RUNX1 may contribute to E2A-HLF-mediated leukemogenesis.

Resistance of infant leukemia with MLL rearrangement to tumor necrosis factor-related apoptosis-inducing ligand: a possible mechanism for poor sensitivity to antitumor immunity.

Malignant cells generally acquire some immune escape mechanisms for clonal expansion. Immune escape mechanisms also contribute to the failure of graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation (allo-SCT). Infant leukemias with mixed-lineage leukemia (MLL) rearrangement have a remarkably short latency, and GVL effect after allo-SCT has not been clearly evidenced in these leukemias. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- and FasL-mediated cytotoxic pathways play important roles in cytotoxic T-lymphocyte- and natural killer cell-mediated antitumor immunity and optimal GVL activity. We investigated the in vitro sensitivity of MLL-rearranged acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) cells to TRAIL- and FasL-mediated cytotoxicity. Most of cell lines and primary leukemia cells were highly resistant to TRAIL primarily owing to low cell-surface expression of death receptors in ALL and simultaneous expression of decoy receptors in AML. Nearly half of cell lines and majority of primary leukemia cells showed low sensitivity to FasL. These results suggest that resistance to death-inducing ligands, particularly to TRAIL, could be one of the mechanisms for a rapid clonal expansion and a poor sensitivity to the GVL effect in infant leukemias with MLL rearrangement.

TGFß1 synergizes with FLT3 ligand to induce chemoresistant quiescence in acute lymphoblastic leukemia with MLL gene rearrangements.

Fms-like tyrosine kinase 3 (FLT3) is highly expressed in mixed-lineage leukemia (MLL) gene-rearranged acute lymphoblastic leukemia (MLL+ALL) with a dismal prognosis. We previously reported that FLT3 ligand (FL) stimulation induced cell cycle arrest in MLL+ALL cells leading to resistance against anti-leukemic agents. Given that FL stimulation enhanced transforming growth factor (TGF)ß1 mRNA levels in MLL+ALL cells, we extensively examined the effect of TGFß1 on the cell cycle progression and chemosensitivity in MLL+ALL cells, and found that TGFß1 stimulation induced MLL+ALL cells into cell cycle arrest resistant to arabinosyl cytosine; its effect was markedly enhanced in synergy with FL. Thus, it is likely that TGFß1 and FL, both abundantly produced by bone marrow stromal cells, function in a coordinated manner to render MLL+ALL cells chemoresistant, which should lead to the development of minimal residual disease (MRD) resulting in relapse. The use of inhibitors against FLT3 and TGFß1 may become a useful strategy for eradicating MRD in MLL+ALL.

Defective control of apoptosis, radiosensitivity, and spindle checkpoint in ataxia telangiectasia.

We examined the regulation of apoptosis, radiosensitivity, and spindle checkpoint in response to DNA-damaging agents in ataxia telangiectasia (AT)-derived lymphoblastoid cell lines (AT-LCLs), which lack AT mutated (ATM) protein expression. In addition to the previous findings that AT-LCLs are defective in regulation of cell cycle at the G1, S, and G2-M checkpoints in response to X-ray irradiation (X-IR) and are highly sensitive to X-IR (J. Biol. Chem., 271: 20486-20493, 1996), we showed for the first time that AT-LCLs were defective in X-IR-associated spindle checkpoint control. The cells were also resistant to early apoptosis as much as LCLs derived from patients with Li-Fraumeni syndrome (LFS-LCLs). Terminal deoxynucleotidyl transferase-mediated nick end labeling assay of LCLs, however, demonstrated a significant increase in apoptotic cells among AT-LCLs cultured over a longer period after X-IR. These findings were in contrast to those of LFS-LCL, which showed very little increase in terminal deoxynucleotidyl transferase-mediated nick end labeling-positive population, even in cells with hyperploidy. Thus, although early apoptosis and cell cycle controls in response to DNA damage are disrupted in both ATM and p53 mutations, cells from AT patients are much more susceptible to late-onset apoptosis than those of LFS. These differences may depend on the level of accumulation of DNA damage and/or threshold that triggers late-onset cell death in ATM or p53 mutations. Our findings allow a better understanding of the role of ATM in p53-dependent and independent signal transduction pathways in response to DNA damaging agents.

DNA damage-associated dysregulation of the cell cycle and apoptosis control in cells with germ-line p53 mutation.

Lymphoblastoid cell lines (LCLs) with heterozygous p53 mutations at residues 286A, 133R, 282W, 132E, and 213ter were established from five independent Li-Fraumeni syndrome families. When cell cycle regulation in response to gamma-irradiation was studied, these LCLs showed an abnormal G1 checkpoint associated with defective inhibition of cyclin E/cyclin-dependent kinase 2 activity in all cases except for 282W LCL, which showed a normal G1 checkpoint. On the other hand, the control of S-phase-G2 as determined by cyclin A/cyclin-dependent kinase 2 activity was defective in all these LCLs. The mitotic checkpoint was also defective in the two LCLs analyzed as either competent or incompetent for G1 arrest. When radiation-induced apoptosis, which requires wild-type p53 function under optimal conditions, was studied, all of these LCLs showed significant failure compared to normal LCLs. These findings indicate that although p53-dependent transactivation and G1-S-phase cell cycle control are variably dysregulated, the induction of apoptosis and control of the cell cycle at S-phase-G2 and the mitotic checkpoint in response to DNA-damaging agents are consistently dysregulated in heterozygous mutant LCLs. This suggests that these dysfunctions underlie, at least in part, the susceptibility of Li-Fraumeni syndrome families to cancer. Furthermore, the approach presented is a potentially useful method for studying individual carriers of different germ-line p53 mutations and different biological features.

Dissociation between cell cycle arrest and apoptosis can occur in Li-Fraumeni cells heterozygous for p53 gene mutations.

The radiation response was investigated in two lymphoblastoid cell lines (LBC) derived from families with heterozygous germ-line missense mutations of p53 at codon 282 (LBC282) and 286 (LBC286), and compared to cells with wt/wt p53(LBC-N). By gel retardation assays, we show that p53-containing nuclear extracts from irradiated LBC282 and LBC286 markedly differ in their ability to bind to a p53 DNA consensus sequence, the former generating a shifted band whose intensity is 30-40% that of LBC-N, the latter generating an almost undetectable band. Unlike LBC286, which fail to arrest in G1 after irradiation, LBC282 have an apparently normal G1/S checkpoint, as they arrest in G1, like LBC-N. While in LBC-N, accumulation of p53 and transactivation of p21WAF1 increase rapidly and markedly by 3 h after exposure to gamma-radiation, in LBC286 there is only a modest accumulation of p53 and a significantly delayed and quantitatively reduced transactivation of p21WAF1. Instead, in LBC282 while p53 levels rise little after irradiation, p21WAF1 levels increase rapidly and significantly as in normal LBC. Apoptotic cells present 48 h after irradiation account for 32% in LBC-N, 8-9% in LBC282 and 5-7% in LBC286, while the dose of gamma-radiation required for killing 50% of cells (LD50) is 400 rads, 1190 rads and 3190 rads, respectively, hence indicating that the heterozygous mutations of p53 at codon 282 affects radioresistance and survival, but not the G1/S cell cycle control. In all LBC tested, radiation-induced apoptosis occurs in all phases of the cell cycle and appears not to directly involve changes in the levels of the apoptosis-associated proteins bcl-2, bax and mcl-1. Both basal as well as radiation-induced p53 and p21WAF1 proteins are detected by Western blotting of FACS-purified G1, S and G2/M fractions from the three cell lines. p34CDC2-Tyr15, the inactive form of p34CDC2 kinase phosphorylated on Tyr15, is found in S and G2/M fractions, but not in G1. However, 24 h after irradiation, its levels in these fractions diminish appreciably in LBC-N but not in the radioresistant LBC286 and LBC282. Concomitantly, p34CDC2 histone H1 kinase activity increases in the former, but not in the latter cell lines, hence suggesting a role for this protein in radiation-induced cell death. Altogether, this study shows that, in cells harbouring heterozygous mutations of p53, the G1 checkpoint is not necessarily disrupted, and this may be related to the endogenous p53 heterocomplexes having lost or not the capacity to bind DNA (and therefore transactivate target genes). Radiation-induced cell death is not cell cycle phase specific, does not involve the regulation of bcl-2, bax or mcl-1, but is associated with changes in the phosphorylation state and activation of p34CDC2 kinase.

Ligand activation of peroxisome proliferator-activated receptor gamma induces apoptosis of leukemia cells by down-regulating the c-myc gene expression via blockade of the Tcf-4 activity.

The peroxisome proliferator-activated receptor gamma (PPAR gamma), a member of the nuclear receptor superfamily, is expressed at highest levels in adipose tissue and functions as a central regulator in the process of adipocyte differentiation. In the present study, we showed that human leukemic cell lines, not only myeloid but also lymphoid, express PPAR gamma and its activation by natural ligand (15-deoxy-Delta(12,14) - prostaglandin J(2)) and synthetic ligand (troglitazone) profoundly inhibited their proliferation by induction of apoptosis preferentially in the serum-free culture. We pursued its mechanism using the representative cell lines, and found that induction of apoptosis was accompanied by caspase-3 activation and specifically blocked by its inhibitor. While status of several apoptosis-related molecules remained unchanged, the c-Myc expression was markedly down-regulated within 24 h after troglitazone treatment. The c-myc mRNA levels were dramatically reduced at 1 h and became undetectable at 12 h after troglitazone treatment, which proved to be accompanied by complete blockade of the Tcf-4 activity in the electrophoretic mobility shift assay. We succeeded in establishing HL-60 cell lines growing well in the presence of troglitazone in the long-term serum-free culture. They showed neither induction of apoptosis nor down-regulation of the c-Myc expression via blockade of the Tcf-4 activity after troglitazone treatment. This is the first identification of the linkage between PPAR gamma-mediated apoptosis and down-regulation of the c-myc gene expression.

Mechanisms of glucocorticoid resistance in human leukemic cells: implication of abnormal 90 and 70 kDa heat shock proteins.

The unliganded glucocorticoid receptor is a multi-oligomer complex consisting of a ligand-binding protein with which two 90 kDa heat shock proteins (hsp90s) are associated. Upon binding of glucocorticoid to the receptor, the ligand-binding protein, which dissociated from hsp90s, enters the nucleus, binds to a specific site in DNA, and thus transmits signal(s). The 70 kDa heat shock protein (hsp70) also works as a molecular chaperone when the ligand-binding protein enters the nucleus. Regarding the mechanisms of glucocorticoid resistance, a decreased expression of glucocorticoid receptor and a mutant protein with low ligand binding affinity have been reported. In the present study, to address other mechanisms of glucocorticoid resistance, we examined the expression of hsp90 and hsp70 in addition to the number of glucocorticoid-binding sites and their affinity using glucocorticoid-sensitive and -resistant human leukemic cell lines. We showed that two of nine resistant cell lines with normal glucocorticoid-binding proteins express aberrant hsp90 and extremely low hsp70, while another seven resistant cell lines had decreased binding sites with normal hsps. These results suggest that there are at least two independent mechanisms of glucocorticoid resistance in human leukemic cell lines: the decreased ligand-binding sites and the abnormal hsps expression.

Expression of thrombopoietin receptor and its functional role in human B-precursor leukemia cells with 11q23 translocation or Philadelphia chromosome.

Thrombopoietin (TPO) is a hematopoietic growth factor which plays a central role in normal megakaryocytopoiesis and thrombopoiesis. Although the interaction between TPO and its receptor c-Mpl encoded by the c-mpl gene is now known to be implicated in the proliferation and/or differentiation of abnormal myeloid cells and normal hematopoietic stem cells, little is known about a role of the TPO/c-Mpl system in lymphoid leukemia cells. In the present study, we first examined the expression of c-mpl/c-Mpl in 23 human lymphoid leukemic cell lines (T-lineage 4, B-lineage 19) using three distinct methods. The c-mpl mRNA was detectable in as many as 20 cell lines (T-lineage 3, B-lineage 17) by reverse transcriptase-polymerase chain reaction, but its translated product, c-Mpl, was demonstrable by Western blot only in B-lineage cell lines. Flow cytometric analysis revealed the surface c-Mpl expression in 13 of 17 B-lineage cell lines, but its higher expression (>40%) was restricted in nine B-precursor cell lines, eight of which had 11q23 translocation or Philadelphia chromosome (Ph1). We also demonstrated that two of eight cell lines with 11q23 translocation or Ph1 exhibited a significant proliferative response to TPO in the 3H-thymidine uptake and colony-forming assays. Triggering of these cell lines by TPO transiently up-regulated tyrosine phosphorylation of JAK-2 and Shc, indicating that their receptor is functional. Primary leukemia cells separated from patients with B-precursor acute lymphoblastic leukemia with Ph1 or 11q23 translocation also showed the surface c-Mpl expression and a significant responsiveness to TPO. These results suggest that the TPO/c-Mpl interaction may play a physiological role in the growth regulation of B-precursor leukemia cells particularly with specific chromosomal abnormalities.

Leukemic cells with 11q23 translocations express granulocyte colony-stimulating factor (G-CSF) receptor and their proliferation is stimulated with G-CSF.

We report a 20-month-old boy with acute lymphoblastic leukemia with the 11q23 translocation whose blasts markedly increased in peripheral blood after intravenous granulocyte colony-stimulating factor (G-CSF) administration, but disappeared after stopping G-CSF. The in vitro study showed that the leukemic cells separated from this patient expressed G-CSF receptor (G-CSFR) and an addition of G-CSF stimulated their proliferation by 3H-thymidine incorporation assay (stimulation index, 4.9). To clarify whether or not leukemic cells with 11q23 translocations generally express G-CSFR and show proliferative response to G-CSF, we performed the similar in vitro experiments using eight leukemic cell lines with 11q23 translocations. We found that all cell lines examined expressed G-CSFR (20-98%) and proliferation of seven leukemic cell lines was significantly enhanced in response to G-CSF (stimulation index >1.5 in five cell lines), suggesting a possible participation of the G-CSF/G-CSFR interaction in the process of growth regulation of leukemic cells with 11q23 translocations.

The JAK2 inhibitor AG490 predominantly abrogates the growth of human B-precursor leukemic cells with 11q23 translocation or Philadelphia chromosome.

The Janus kinase (JAK) family is one of intracellular protein tyrosine kinases (PTKs) present in hematopoietic and lymphoid cells and has been shown to play a crucial role in a variety of biological responses. It was reported that a human B-precursor leukemic cell line was potently inhibited in its proliferation by one of synthetic PTK inhibitors (tyrphostins), AG490, via anti-JAK2 activity. However, no extensive studies about it have been performed. In the present study, we tested 16 human lymphoid leukemic cell lines (B-precursor, 12; T cell, four) for their sensitivity to AG490 using 3H-thymidine incorporation and colony formation assays, and found that B-precursor cell lines with 11q23 translocation or Philadelphia chromosome (Ph1) whose JAK2 proved to be constitutively phosphorylated were predominantly sensitive to AG490 at a concentration that has few inhibitory effect on normal hematopoiesis. We first revealed the association of JAK2 with BCR-ABL in Ph1-positive cell lines and with Bruton's tyrosine kinase (BTK) in cell lines with 11q23 translocation by coimmunoprecipitation experiments. Of interest, AG490 markedly down-regulated phosphorylation of JAK2, but rather transiently up-regulated phosphorylation of BCR-ABL and BTK, suggesting direct implication of AG490 in the process of the JAK2 dephosphorylation. These results indicate that AG490 exerts a potent inhibitory activity to B-precursor leukemia with specific chromosomal abnormalities, and a therapeutic approach using AG490 is expected.

Participation of granulocyte colony-stimulating factor in the growth regulation of leukemia cells from Philadelphia chromosome-positive acute leukemia and blast crisis of chronic myeloid leukemia.

Granulocyte colony-stimulating factor (G-CSF) has been shown to support the growth of multipotential hematopoietic stem cells in addition to the cells of neutrophilic lineage. Philadelphia chromosome (Ph1)-positive leukemia has its origin in the hematopoietic stem cell. In the present study, we demonstrated that the proliferation of leukemic cells from chronic myeloid leukemia in blast crisis (CML-BC) and Ph1-positive acute lymphoblastic leukemia (ALL) cases is frequently stimulated with G-CSF in vitro. We next studied a total of 12 leukemic cell lines established from CML-BC (n= 6) and Ph1-positive acute leukemia (n= 6): four 'myeloid', five 'biphenotypic', and three 'lymphoid' types. All cell lines expressed G-CSF receptor (G-CSFR) in flow cytometric analysis, but their proliferative response to G-CSF in 3H-thymidine incorporation assay varied. The 'biphenotypic' cell lines expressed G-CSFR at higher levels and showed the most pronounced response to G-CSF. The 'lymphoid' cell lines showed intermediate G-CSFR expression with the modest response to G-CSF. Unexpectedly, 'myeloid' cell lines showed lower G-CSFR expression and lower G-CSF response compared with 'biphenotypic' cell lines. In three of four 'myeloid' cell lines, proliferation was partially inhibited by an addition of anti-G-CSF neutralizing monoclonal antibody into culture medium. Further, the % inhibition of 3H-thymidine uptake of cell lines positively correlated with the amount of their intracellular G-CSF measured by enzyme immunoassay, suggesting an autocrine growth mechanism via the G-CSF/G-CSFR interaction. These results suggest that G-CSF play an important role in the growth regulation of leukemia cells from Ph1-positive acute leukemia and CML-BC.

The KOR-SA3544 antigen predominantly expressed on the surface of Philadelphia chromosome-positive acute lymphoblastic leukemia cells is nonspecific cross-reacting antigen-50/90 (CD66c) and invariably expressed in cytoplasm of human leukemia cells.

We previously reported a novel monoclonal antibody KOR-SA3544 which predominantly reacted with a surface antigen (sSA3544) expressed on Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukemia (ALL). In the present study, we demonstrate that the antibody specifically recognized nonspecific cross-reacting antigen (NCA)-50/90 (CD66c), one of the carcinoembryonic antigen (CEA)-related glycoproteins encoded by a member of the CEA gene family. In addition, we show that the SA3544 antigen (NCA-50/90) was invariably expressed in cytoplasm of all of the human leukemic cell lines examined (sSA3544-positive B-lymphoid two, sSA3544-negative T or B-lymphoid and non-lymphoid 24) regardless of the presence or absence of surface expression of this antigen. Immunoelectromicroscopic examination revealed that the cytoplasmic antigen was mainly present in granules in sSA3544-positive leukemia cells, whereas it was diffusely present in cytosol in sSA3544-negative leukemia cells. Thus, among members of the CEA family, NCA-50/90 was first demonstrated to be expressed not only on the surface of some leukemia cells, but also in cytoplasm of various types of leukemia cells.

p16/MTS1/INK4A gene is frequently inactivated by hypermethylation in childhood acute lymphoblastic leukemia with 11q23 translocation.

The p16 gene encoding a specific inhibitor of cyclin-dependent kinases 4 and 6 has been reported to be inactivated at a variety of rates in malignant tumors. We studied frequency and mechanism of inactivation of the p16 gene in various types of childhood acute lymphoblastic leukemia (ALL) using 36 leukemic cell lines established from children (B precursor-ALL, 28; B-ALL/Burkitt's lymphoma, 3; and T-ALL, 5). On Southern blot, homozygous deletions or hemizygous deletions with rearrangement were detected in 14 cell lines. The expression of p16 protein was not observed on Western blot in 18 of 22 cell lines with intact p16 gene, but induced in 16 cell lines after treatment with the demethylating agent, indicating the silencing of the p16 gene by hypermethylation. Of note, the p16 gene was inactivated by hypermethylation of the 5' CpG island in nine of nine cell lines with 11q23 translocation, but was restored with the treatment of the demethylating agent. Partial methylation of the p16 gene was also demonstrated in three of eight primary leukemia samples with this translocation, suggesting that the p16 gene inactivation by hypermethylation might play a role in the leukemogenesis and disease progression of ALL with 11q23 translocation.

Flow cytometric functional analysis of multidrug resistance by Fluo-3: a comparison with rhodamine-123.

Using four cell lines including drug-sensitive K562/Parent cells, P-glycoprotein (Pgp)-mediated multidrug resistant (MDR) K562/VCR, K562/ADR and revertant K562/ADR-R cells, two fluorescent agents, Fluo-3 and rhodamine-123 (Rh-123), were compared as indicators in a functional assay of MDR. Cells were incubated with 4 microM Fluo-3 or 1 microM Rh-123 for 45 min and then the intracellular accumulation of the agent was measured using a flow cytometer. Verapamil (20 microM) or cepharanthine (biscoclaurine alkaloid, 10 microM) was added just before the fluorescent agents. Efflux patterns were also studied 60 min after incubation with or without verapamil and cepharanthine. Increased intracellular accumulation and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were demonstrated in multidrug resistant K562/VCR and K562/ADR cells, indicating that Fluo-3 is another good indicator of MDR. However, a similar, but lower, increase in uptake and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were also demonstrated even in Pgp-non-overexpressed K562/Parent cells. In contrast, accumulation of Rh-123 was not affected by verapamil and cepharanthine. To further study the Pgp dependency of Fluo-3, another cell line, K562/NC16 expressing minimum MDR1 mRNA, was cloned. Increased uptake and a delayed efflux pattern of Fluo-3, but not Rh-123, with verapamil or cepharanthine were again demonstrated in K562/NC16 cells, indicating that intracellular accumulation of Fluo-3 may be non-specifically influenced by verapamil and cepharanthine at very low levels of Pgp-related MDR, while the influx and efflux patterns of Rh-123 may be specifically affected by Pgp overexpression.

Analysis of serum lactate dehydrogenase and its isozymes during administration of granulocyte colony-stimulating factor in children.

Serum lactate dehydrogenase (LDH) activity is known to become elevated following granulocyte colony-stimulating factor (G-CSF) therapy in some patients, but no extensive studies on this phenomenon have been performed. In 26 children with malignancies in complete remission who received recombinant human G-CSF intravenously after combined chemotherapy, we measured serum LDH and its isozymes (81 episodes) before chemotherapy (pre-Tx), during administration of G-CSF (mid-Tx), and after stopping G-CSF (post-Tx) and compared the obtained data using paired t test. Twelve episodes were excluded from analysis because of liver dysfunction (alanine aminotransferase > 45 IU/L). Serum LDH at mid-Tx (343.1+/-19.8 IU/L; mean +/- SE) was significantly higher than that at pre-Tx (186.9+/-6.7 IU/L) and post-Tx, but this difference was observed only when change in white blood cell counts (WBCs) (WBC at mid-Tx minus WBC at pre-Tx) was greater than or equal to 4000/microL (58 episodes). Percentages of LDH isozymes 3, 4, and 5 at mid-Tx (23.5+/-1.0, 11.7+/-0.8, and 8.3+/-0.7) were significantly increased compared with those at pre-Tx (19.5+/-0.7, 6.3+/-0.3, and 4.2+/-0.5) and post-Tx, resulting in a significant decrease in percentages of LDH isozymes 1 and 2 at mid-Tx. In episodes of change in WBCs > or =4000/microL, change in LDH significantly correlated to changes in WBCs and granulocytes but not to change in lymphocytes or monocytes. These results suggest that serum LDH is significantly elevated during G-CSF administration in accordance with the increase in peripheral granulocytes, which accompanies change in the pattern of percentages of LDH isozymes.

Clonal hematopoiesis in acquired aplastic anemia revealed by rearrangement of the immunoglobulin heavy chain gene-JH region.

We report on a female patient with acquired aplastic anemia whose bone marrow cells showed DNA rearrangement of the immunoglobulin-JH region that disappeared after 1 month with recovery of hematopoiesis through treatment with granulocyte colony-stimulating factor (G-CSF) and immunosuppressive drugs. The patient is now 2 years and 6 months from onset, and her hematopoiesis is almost within normal limits without medication. This finding provides new data supporting clonal hematopoiesis in acquired aplastic anemia but does not imply that the disease is resistant to immunosuppressive therapy.

Development of acute lymphoblastic leukemia with translocation (4;11) in a young girl with familial pericentric inversion 12.

We report a case of a 1-year-old girl with familial pericentric inv(12) who developed acute lymphoblastic leukemia (ALL) with t(4;11) 1 month after recovery from idiopathic hemophagocytic lymphohistiocytosis (HLH). The inv(12)(p13q15) was first found in bone marrow (BM) cells when she was diagnosed as having HLH, and then detected in the BM blasts together with t(4;11)(q21;q23) when she developed ALL. The inv(12) was retained in the BM cells after she achieved complete remission. Cytogenetic analysis on the PHA-stimulated peripheral lymphocytes revealed inv(12) in all of the 30 cells examined. Because the data that ALL with t(4;11) predicts an extremely poor prognosis, she received an allogeneic BM transplantation from an HLA-matched sibling at 10 months from the onset of ALL. She is now at 26 months post transplantation and maintains in a state of complete remission. Familial cytogenetic study demonstrated that 4 of 8 maternal members examined had the inv(12), but they showed no family history of a higher risk of development of hematological and other types of malignancies, suggesting that pericentric inv(12) itself might not be directly involved in the development of ALL in this case.