Rac1 signaling protects monocytic AML cells expressing the MLL-AF9 oncogene from caspase-mediated apoptotic death.

We investigated the relevance of signaling mechanisms regulated by the Ras-homologous GTPase Rac1 for survival of acute myeloid leukemia (AML) cells harbouring the MLL-AF9 oncogene due to t(9;11)(p21;q23) translocation. Monocytic MLL-AF9 expressing cells (MM6, THP-1) were hypersensitive to both small-molecule inhibitors targeting Rac1 (EHT 1864, NSC 23766) (IC50EHT ~12.5 µM) and lipid lowering drugs (lovastatin, atorvastatin) (IC50Lova ~7.5 µM) as compared to acute myelocytic leukemia (NOMO-1, HL60) and T cell leukemia (Jurkat) cells (IC50EHT >30 µM; IC50Lova >25 µM). Hypersensitivity of monocytic cells following Rac1 inhibition resulted from caspase-driven apoptosis as shown by profound activation of caspase-8,-9,-7,-3 and substantial (~90 %) decrease in protein expression of pro-survival factors (survivin, XIAP, p-Akt). Apoptotic death was preceded by S139-posphorylation of histone H2AX (γH2AX), a prototypical surrogate marker of DNA double-strand breaks (DSBs). Taken together, abrogation of Rac1 signaling causes DSBs in acute monocytic leukemia cells harbouring the MLL-AF9 oncogene, which, together with downregulation of survivin, XIAP and p-Akt, results in massive induction of caspase-driven apoptotic death. Apparently, Rac1 signaling is required for maintaining genetic stability and maintaining survival in specific subtypes of AML. Hence, targeting of Rac1 is considered a promising novel strategy to induce lethality in MLL-AF9 expressing AML.

Regulated cellular exposure to non-thermal plasma allows preferentially directed apoptosis in acute monocytic leukemia cells.

This research investigated the modulation of cell death through exposure of non-thermal resistive barrier based indirect air plasma on monocytic leukemia cancer cells (THP-1). Specifically, we explored cell death through apoptosis and necrosis, since generally apoptotic cell death has a limited inflammatory response as compared to necrosis. We have demonstrated a preference for apoptosis in plasma treated THP-1 cells, under specific plasma characteristics and dosage levels, using fluorescent dyes conjugated with annexin V followed by identification of the cells through fluorescent microscopy and flowcytometry diagnostics. At much higher plasma dosages, the necrotic morphologies in the THP-1 cells were observed. The presented outcomes in the death morphologies of plasma treated THP-1 cells signify the need for further investigation on the cellular mechanisms induced by the indirect plasma exposure. The results obtained from this research indicate the significant potential for the use of our portable non-thermal resistive barrier based indirect plasma treatment method as an inexpensive and less invasive method for treating leukemia and other cancerous lesions.

Characterization of an atmospheric pressure plasma jet and its applications for disinfection and cancer treatment.

In this work an atmospheric pressure non-thermal resistive barrier (RB) plasma jet was constructed, characterized and was applied for biomedical applications. The RB plasma source can operate in both DC (battery) as well as in standard 60/50 Hz low frequency AC excitation, and it functions effectively in both direct and indirect plasma exposure configurations. The characteristics of the RB plasma jet such as electrical properties, plasma gas temperature and nitric oxides concentration were determined using voltage-current characterization, optical emission spectroscopy and gas analyzer diagnostic techniques. Plasma discharge power of 26.33 W was calculated from voltage-current characterization. An optical emission spectroscopy was applied and the gas temperature which is equivalent to the nitrogen rotational (Trot) temperatures was measured. The concentrations of the reactive oxygen species at different spatial distances from the tip of the plasma jet were measured and the ppm concentration of NO is at the preferred level for a wide range of standard biomedical treatment applications. The ppm values of nitric oxides after the cooling unit are observed to be of the same order of magnitude as compared to plasma jet. The portable RB plasma source was tested to be very effective for decontamination and disinfection of a wide range of foodborne and opportunistic nosocomial pathogens such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus cereus and the preliminary results are presented. The effects of indirect exposure of the portable RBP source on monocytic leukemia cancer cells (THP-1) were also tested and the results demonstrate that a preference for apoptosis in plasma treated THP-1 cells under particular plasma parameters and dosage levels.

Identification and functional characterization of the novel acute monocytic leukemia associated antigen MLAA-34.

We have previously applied the method of serologic analysis of recombinant cDNA expression library (SEREX) on acute monocytic leukemia to identify monocytic leukemia-associated antigens. Using this approach, we identified a novel gene, MLAA-34, which exclusively reacted with sera from allogeneic leukemia patients but not with normal donor sera. Here, we further characterized its gene structure and explored the function. We first determined both 5' and 3' end by RLM-RACE and cloned full-length cDNA of MLAA-34 in U937 cell line. Analysis of full cDNA sequence showed that MLAA-34 is highly homologous to known human gene CAB39L, but differs from two transcript splice variants of CAB39L. Thus, we propose that MLAA-34 is a novel CAB39L's splice variant associated with acute monocytic leukemia. Because the functions of MLAA-34 and CAB39L are both very unclear, then we investigated the role of MLAA-34 in U937 cell line using RNA interference technology. The results showed that the downregulation of MLAA-34 expression significantly suppressed the proliferation of U937 cells in vitro, and increased the spontaneous apoptosis of these leukemia cells. All these data indicated that MLAA-34 may be a novel anti-apoptotic factor related closely to carcinogenesis or progression of acute monocytic leukemia. The anti-apoptotic pathways of MLAA-34 remain further exploration. This study warrants further investigations to verify MLAA-34 as a promising antigen and a molecular target for therapeutic applications in acute monocytic leukemia.

Ocular infiltration as initial presentation of acute monocytic leukaemia transformed from chronic myelomonocytic leukaemia associated with BRAF V600E mutation.

Acute monocytic leukaemia (French-British-American classification: AML-M5b) is characterised by a predominance of cells of the monocytic lineage on bone marrow examination. Furthermore, a discerning feature is its tendency for tissue infiltration. While gum hypertrophy and hepatosplenomegaly are common, ocular involvement is rare. Here, we present a case of a 75-year-old man referred with proptosis and monocytosis-subsequently diagnosed as AML-M5b, whose disease course was distinguished by extensive tissue invasion (ocular, pulmonary, liver, spleen). Cytogenetics and molecular tests were consistent with blastic transformation of previously undiagnosed chronic myelomonocytic leukaemia, supported by the presence of long-standing, low-grade monocytosis. Notably, a BRAF V600E mutation was also detected-an oncogenic driver previously reported in de novo and therapy-related, but not chronic myelomonocytic leukaemia-transformed, AML-M5b. While an initial response to cytoreductive treatment was observed, his tissue-invasive disease soon progressed with worsening pulmonary infiltrates, disseminated intravascular coagulation and renal failure, resulting in death.

Evolution of tigecycline- and colistin-resistant CRKP (carbapenem-resistant Klebsiella pneumoniae) in vivo and its persistence in the GI tract.

Emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains that also exhibit resistance to tigecycline and colistin have become a major clinical concern, as these two agents are the last-resort antibiotics used for treatment of CRKP infections. A leukemia patient infected with CRKP was subjected to follow-up analysis of variation in phenotypic and genotypic characteristics of CRKP strains isolated from various specimens at different stages of treatment over a period of 3 years. Our data showed that (1) carbapenem treatment led to the emergence of CRKP in the gastrointestinal (GI) tract of the patient, which subsequently caused infections at other body sites as well as septicemia; (2) treatment with tigecycline led to the emergence of tigecycline-resistant CRKP, possibly through induction of the expression of a variant tet(A) gene located in a conjugative plasmid; (3) colistin treatment was effective in clearing CRKP from the bloodstream but led to the emergence of mcr-1-positive Enterobacteriaceae strains as well as colistin-resistant CRKP in the GI tract due to inactivation of the mgrB gene; and (4) tigecycline- and colistin-resistant CRKP could persist in the human GI tract for a prolonged period even without antibiotic selection pressure. In conclusion, clinical CRKP strains carrying a conjugative plasmid that harbors the blaKPC-2 and tet(A) variant genes readily evolve into tigecycline- and colistin-resistant CRKP upon treatment with these two antibiotics and persist in the human GI tract.

Autocrine proliferative effect of ghrelin on leukemic HL-60 and THP-1 cells.

Ghrelin is a 28 amino acid peptide hormone that is mainly produced by the stomach, but also by several tissues and tumors. Ghrelin is octanoylated on the Ser(3), but is also detected as a des-acylated form. Only the acylated ghrelin activates the GH secretagogue receptor (GHS-R) type 1a to stimulate GH release, and regulate food intake and energy metabolism. For the first time, we report that ghrelin and des-acyl ghrelin are present in human promyelocytic HL-60, monocytic THP-1 and lymphoblastic SupT1 cell lines. The human leukemic cell lines did not express the functional GHS-R 1a, whereas they expressed GHS-R 1b, a truncated variant of the receptor. Leukemic cell proliferation was not modified by the addition of octanoylated or des-acyl ghrelins. However, THP-1 and HL-60 cell proliferations were inhibited by SB801, an antibody directed against the N-terminal octanoylated portion of ghrelin, suggesting that octanoylated ghrelin stimulates cell proliferation via an autocrine pathway involving an as yet unidentified ghrelin receptor. Both octanoylated and des-acyl ghrelins did not alter the basal adenylate cyclase activity. Treatments of THP-1 and SupT1 cells by both octanoylated and des-acyl ghrelins did not modify the adenylate cyclase activity in response to vasoactive intestinal peptide, suggesting that ghrelin is unlikely to modulate the anti-inflammatory and differentiating properties of vasoactive intestinal peptide.

Alpha (p55) and beta (p75) chains of the interleukin-2 receptor are expressed by AML blasts.

In the present study, we have investigated the leukemic cells obtained from 16 patients with acute myeloid leukemia (AML) at diagnosis for the membrane expression of p55 (alpha) and p75 (beta) interleukin-2 receptor (IL-2R) chains using specific monoclonal antibodies (mAbs), as well as for the presence of their transcripts using Northern blot analysis. In addition, immunoprecipitation of the p75 membrane molecule with TU27 and Mik-beta 1 mAbs was carried out in selected cases. The p75 IL-2R beta transcripts were detected in all cases, whereas the membrane p75 molecule was demonstrable by flow cytometry in three cases. However, data from the immunoprecipitation analysis suggest that the lack of the p75 IL-2R detection by flow cytometry might be caused by the low density of molecules per cell rather than the fact that the specific mRNA is not translated into the p75 surface molecule. In addition, a consistent membrane positivity with an anti-p55/CD25 mAb, present on fresh uncultured blasts in 37.5% of the cases, became detectable after short-term culture in 75% of cases. In each individual case, a strict correlation was found between membrane CD25 reactivity and the expression of p55 mRNA. Taken together, our data suggest that the expression of both alpha (p55) and beta (p75) IL-2R molecules is a common feature of leukemic cells in AML, and provide new arguments for reassessing the possible role of IL-2 in leukemic growth.

Stimulation of diploid fibroblast growth with serum-free medium conditioned by mezerein-treated monocytic leukemia cells.

Medium conditioned by mezerein-treated human acute monocytic leukemia cells (THP-1) stimulated human fibroblast replication. Maximum mitogenic activity was elaborated by THP-1 cells with a 24-hr incubation in 10(-7) M mezerein (activator phase) followed by a 36-hr incubation in insulin-supplemented serum-free Roswell Park Memorial Institute (RPMI)-1640 medium (effector phase). Growth stimulation was not due to the presence of residual mezerein. We previously reported that leukemia cells also produced a growth inhibitor. Fibroblast stimulation was resolved by isoelectrofocusing into several active fractions separate from the growth inhibitory activity for malignant mammary cells. Conditioned medium was mitogenic for fibroblasts in the presence of high concentrations of fetal bovine and human whole blood sera. Growth stimulation was observed in plasma-derived serum only when supplemented with exogenous platelet-derived growth factor. Thus, this THP-1 cell product does not fulfill the role of a competence factor.

Cellular and cytokine dependent monocyte-mediated leukemic cell death: modulation by interferon-gamma and tumor necrosis factor-alpha.

Activated human monocytes and macrophages are involved in host defense against neoplastic cells. In view of cellular adoptive immunotherapy, we have studied the role of tumor necrosis factor-alpha (TNF-alpha) and gamma-interferon (IFN-gamma) in monocyte-mediated cytotoxicity on the level of both effector and leukemic target cells. Highly purified and IFN-gamma-activated monocytes were cytolytic to U937 cells up to 81.9 +/- 5.3% (mean +/- SEM) in a 24-hour MTT cytotoxicity assay at an effector-to-target-cell ratio of 10. Upon IFN-gamma activation these monocytes showed a 20-fold increase in TNF-alpha secretion of 663 +/- 122 pg/mL. Comparable concentrations of recombinant human TNF-alpha showed only cytostatic effects on U937 cells of approximately 20% after 24 hours, similar to the cytostatic effects of IFN-gamma-activated monocyte culture supernatants. These effects could be fully reversed by anti-TNF-alpha antibodies. U937 cells pretreated with TNF-alpha were almost completely resistant to monocyte-mediated cytotoxicity, supernatant-mediated cytostasis and to TNF-alpha up to 10(4) U/mL. IFN-gamma-activated monocytes were able to lyse TNF-alpha-modified U937 cells whereas IFN-gamma-activated monocyte supernatants showed only cytostatic activity after prolonged incubation. Additionally, target cell modulation by IFN-gamma potentiated the TNF-alpha-dependent cytolytic and cytostatic effects of monocytes, monocyte culture supernatants and TNF-alpha. We conclude that monocytes as a cellular component in monocyte-mediated cytotoxicity are far more potent in lysis of leukemic target cells than are secreted monokines. Furthermore, IFN-gamma and TNF-alpha are involved in the regulation of the susceptibility of leukemic cells for lysis by interactions with monocytes.

Infiltrative polyneuropathy due to acute monoblastic leukemia in hematologic remission.

A 66-year-old man with acute monoblastic leukemia developed acute polyneuropathy with quadriplegia, autonomic instability, and respiratory failure while he was in hematologic remission following both systemic and intrathecal chemotherapy. Autopsy revealed dense infiltration of somatic and autonomic peripheral nerves, sparing the meninges. There was a small peripheral infiltrate in one of four dorsal root ganglia, but, otherwise, sensory and autonomic ganglia were normal. The blood-nerve barrier may allow some malignant cells to escape cytotoxic agents. The epineurium and ganglia lack a blood-tissue barrier, and malignant cells could have been eradicated at those sites.

The effect of a synthetic 7-thiaprostaglandin E1 derivative, TEI-6122, on monocyte chemoattractant protein-1 induced chemotaxis in THP-1 cells.

1 The ability of various prostaglandins (PGs) to inhibit monocyte chemotaxis induced by monocyte chemoattractant protein-1 (MCP-1) was investigated with a human monocytic leukaemia cell line, THP-1. Moreover, to investigate the mechanism of the inhibitory action of PGs the involvement of either intracellular adenosine 3': 5'-cyclic monosphosphate (cyclic AMP) accumulation or intracellular Ca2+ mobilization was studied. 2 TEI-6122, a synthetic 7-thia-PGE1 derivative, inhibited chemotaxis of THP-1 cells induced by MCP-1 with an IC50 of 1.5 pM. Its inhibitory activity was 1000 fold more than that of PGE1 and PGE2 (IC50 = 2.8 nM and 0.9 nM, respectively), which were more potent than other PGs such as PGA1, PGA2, PGF2 alpha and PGI2 (IC50 > or = 1 microM). 3 With respect to the effect on intracellular cyclic AMP accumulation in THP-1 cells, TEI-6122 was as potent as PGE1 and PGE2, which were approximately 100 to 1000 fold more potent than the other PGs such as PGA1, PGA2 and PGI2. The minimum concentration of TEI-6122 required to increase intracellular cyclic AMP accumulation in THP-1 cells was 1 nM. 4 TEI-6122 and PGE1 (4 microM) transiently increased intracellular calcium levels in THP-1 cells. When added prior to MCP-1, both PGs partially suppressed the increased in Ca2+ caused by this cytokine. There were no significant differences between the activity of TEI-6122 and PGE1 in either respect. 5 It is concluded that TEI-6122, a synthetic 7-thia-PGE1 derivative is a much more potent inhibitor of MCP-1-induced THP-1 cell chemotaxis than PGEI and PGE2 which are the best inhibitors among the natural PGs tested, while neither intracellular cyclic AMP accumulation nor effects on Ca2+ mobilization account for the extremely potent inhibitory activity of TEI-6122. Thus, either a novel PGE2 receptor (EPreceptor) or a novel intracellular signal transduction system may be involved in the extremely potent chemotaxis inhibitory activity of TEI-6122.

Pure acute monocytic leukemia. A study of 12 cases.

Twelve cases of pure acute monocytic leukemia in adults were studied. They were selected on the basis of the morphology of the blast cells on Romanowsky-stained smears of blood and bone marrow, as well as positivity of the cells for the naphthol ASD acetate esterase reaction specifically inhibited by sodium fluoride. There was no sex predominance. Neoplastic involvement of the skin and/or gingiva was very frequent. The leukemic proliferation in blood and bone marrow consisted of monoblasts, promonocytes and monocytes. The peroxidase reaction was negative or only faintly positive. Serum and urinary lysozyme levels were increased. The blast cells retained their ability to stimulate, in vitro, colony formation by normal bone marrow cells used as targets. All of these characteristics permit specific identification of this type of acute leukemia. The prognosis is grim: only five of 12 patients achieved complete remission, and four of these five had relapses in less than 14 months; the median survival was five months.

Histiocytic sarcomas and monoblastic leukemias. A clinical, histologic, and immunophenotypical study.

Eight histiocytic sarcomas, identified by examination of more than 2000 malignant lymphomas, are described. For comparison, tumor infiltrates from five monoblastic leukemias were also analyzed. The histiocytic sarcomas were all high-grade malignancies consisting of markedly pleomorphic large cells with many mitotic figures. At presentation, six of the patients had systemic symptoms (fever, fatigue, loss of weight), skin infiltrates, and lymphadenopathy. Despite aggressive chemotherapy, clinical remissions were short, and six patients died of disease .5-48 months (mean, 6.5 months) after diagnosis. The remaining two patients are alive and in partial or complete remission 7 and 12 months after diagnosis. Immunotypic examination showed that all the histiocytic sarcomas were positive for macrophage-related antigens and negative for antigens on B cells, T cells, myeloid cells, epithelial cells, and melanocytes. T-cell receptor and immunoglobulin genes were studied in three cases and were present in a germline configuration. One of the histiocytic sarcomas resembled Langerhans' cells in phenotype and morphology; it was classified as a Langerhans' cell sarcoma. The remaining histiocytic sarcomas did not express accessory cell-associated antigens, but more closely resembled "ordinary" tissue macrophages; they were positive for lysozyme and/or CD68, followed in frequency by CD11c, CD4, CD11b, CDw32, peanut agglutinin receptor, and CD13. Similar features were seen in the monoblastic leukemias. These conditions could only be distinguished from histiocytic sarcoma by clinical and morphologic, rather than immunophenotypic, criteria. Expression of oncoprotein p53 was studied in nine cases and was positive in six of six histiocytic sarcomas and one of three monoblastic leukemias. Rare malignancies show features consistent with the derivation from macrophages. Two entities may be distinguished: those that resemble antigen-presenting accessory cells and those that more closely resemble ordinary tissue macrophages. Recognition of these tumors is important clinically and requires assessment of clinical, morphologic, and immunophenotypic features, supplemented by analysis of T-cell receptor and immunoglobulin genes. Whether (or how) p53 gene mutations are implicated in their pathogenesis will be an important topic for future investigation.

Disappearance of hypotetraploid clones after short-term culture of leukemic cells. A case report.

A case of M4-AML (acute monoblastic leukemia) is presented which was found to be hypotetraploid (79-80-? chromosomes per metaphase) with direct chromosome preparation while 24- and 48-hour cultures contained predominantly normal and some nonclonal hyperdiploid cells. This observation emphasizes the need for both direct and culture methods to disclose the true karyotype of leukemic cells.

Translocation (16;21)(p11;q22) in acute monoblastic leukemia with erythrophagocytosis.

A patient with acute monoblastic leukemia with erythrophagocytosis and a t(16;21) (p11;q22), poor response to chemotherapy, early relapse, and a short survival of ten months is presented. Hematologically, this patient could be considered as a case of FAB M5b/t(8;16) but without the characteristic chromosomal translocation, i.e., there is no visible alteration on chromosome 8 and the breakpoint on chromosome 16 appears to be very proximal. These findings are briefly discussed in the light of other variants.

Acute monocytic leukemia: a single institution experience.

Using strict FAB criteria, 39 cases of monocytic leukemia were identified in 463 consecutive cases of AML. Patients had a median age of 49 with no sex predominance. Extramedullary disease and hyperleukocytosis were common (54% and 36% of patients respectively). Cytogenetic analysis was successful in 38 of 39 patients; 71% had a cytogenetic abnormality and 42% of these involved chromosome 11; 14 of 16 chromosome 11 abnormalities involved the region of 11q23. Non-chromosome 11 abnormalities tended to occur in older patients and to be associated with a lower platelet count; patients with the translocation 9;11 tended to have a lower white count and a higher incidence of therapy-related leukemia. 35 patients were treated with induction therapy including intensive chemotherapy (n = 33) and allogeneic BMT at presentation (n = 2). Patients who entered remission underwent consolidation chemotherapy, autologous BMT, or allogeneic BMT depending on policies at the time of diagnosis. Of 6 patients who underwent further intensive chemotherapy there is 1 long-term disease-free survivor. 3 of 8 patients undergoing autologous BMT and 2 of 3 patients undergoing allogeneic BMT are long-term disease-free survivors. We conclude that this specific subtype of AML, relatively rare when strict criteria are applied, is associated with unique biologic and clinical features and that the high relapse rate associated with conventional therapy makes new treatment approaches involving stem cell transplantation or immunomodulation necessary.

[Morphofunctional characteristics of the blast cells in acute myelomonoblastic leukemia]. / Morfofunktsional'nye osobennosti blastnykh kletok pri ostrom mielomonoblastnom leikoze.

Morphocytochemical and immunological characteristics and indices of the cloning effectiveness were studied in agar cultures. Three types of leukemic cells were found whose ratio in the bone marrow varied in a rather wide range. The lymphoid markers (of cOLL and T-antigens) were detected on blast cells. It is established that histogenetic characteristics of the blast cells determines peculiarities of their growth in the agar culture.

Clinicopathologic aspects of acute leukemias in the dog.

Nineteen cases of canine acute leukemia were diagnosed during a 4-year period. Two main categories were identified on the basis of cytologic, hematologic, and clinical features: acute lymphoid leukemia and acute myelogenous leukemia. Clinical features included history of weight loss, anorexia, shifting limb lameness, and incoordination. Physical findings were characterized by hepatomegaly, splenomegaly, mild generalized lymphadenopathy, and pallor. Ocular lesions were found in 29% of dogs with acute myelogenous leukemia. Hematologic abnormalities included anemia, thrombocytopenia, pancytopenia, leukemia, and leukoerythroblastic reactions. Results of therapy were discouraging.

[Expression of NM23-H(1) mRNA in acute leukemia].

OBJECTIVE: To determine the expression of NM23-H(1) gene in acute leukemia(AL) and evaluate the relationship between NM23-H(1) expression and clinical features. METHODS: Expression level of NM23-H(1) mRNA in bone marrow cells was determined in 82 acute leukemia patients and 15 normal subjects with semi-quantitative reverse transcription polymerase chain reaction(RT-PCR). RESULTS: NM23-H(1)/ GAP DH ratio >/= 0.5 was considered to be positive. NM23-H(1) mRNA was negative in all the 15 normal subjects. Expression of NM23-H(1) was positive in 43 of the 56 acute leukemia patients in the first visit, expression range being 0.33 approximately 2.75. There was one positive case in 12 AL patients with complete remission, expression range being 0 approximately 0.63,but there was no positive case in 6 AL patients who had maintained complete remission for more than 6 months, expression range being 0 approximately 0.27. Relapsed cases were all positive with an expression range of 0.76 approximately 1.87. NM23-H(1) expression in patients with initial and relapsed acute leukemia was higher than that in normal subjects (P < 0.01). CONCLUSION: Overexpression of NM23-H(1) mRNA can predict treatment outcome and may be an important prognostic factor.