Inter-observer reproducibility of endometrial cytology by the Osaki Study Group method: utilising the Becton Dickinson SurePath™ liquid-based cytology.

OBJECTIVE: The purpose of the present study was to evaluate the reproducibility of the cytological diagnosis of endometrial lesions by the Osaki Study Group (OSG) method of new cytological diagnostic criteria using BD SurePath™ (SP)-liquid-based cytology (LBC). METHODS: This cytological classification using the OSG method consists of six categories: (i) normal endometrium (NE), (ii) endometrial glandular and stromal breakdown (EGBD), (iii) atypical endometrial cells, cannot exclude atypical endometrial hyperplasia or more (ATEC-A), (iv) adenocarcinoma including atypical endometrial hyperplasia or malignant tumour (Malignancy), (v) endometrial hyperplasia without atypia (EH) and (vi) atypical endometrial cells of undetermined significance (ATEC-US). For this study, a total 244 endometrial samplings were classified by two academic cytopathologists as follows: 147 NE cases , 36 EGBD cases , 47 Malignant cases, eight ATEC-A cases, two EH cases and four ATEC-US cases. To confirm the reproducibility of the diagnosis and to study the inter- and intra-observer agreement further, a second review round followed at 3-month intervals, which included three additional cytopathologists. RESULTS: The inter-observer agreement of NE classes improved progressively from 'good to fair' to 'excellent', with values increasing from 0.70 to 0.81. Both EGBD and Malignancy classes improved progressively from 'good to fair' to 'excellent', with values increasing from 0.62-0.63 to 0.84-0.95, respectively. The overall intra-observer agreement between the first and the second rounds was 'good to fair' to 'excellent', with values changing from 0.79 to 0.85. All kappa improvements were significant (P < 0.0001). CONCLUSION: In this study, it seemed that the use of the OSG method as the new diagnostic criteria for SP-LBC preparation, may be a valid method to improve the precision (reproducibility) of endometrial cytology.

WT1 immunoenzyme staining using SurePath(™) processed urine cytology helps to detect kidney disease.

OBJECTIVES: Damage and detachment of podocytes and loss into the urine have been implicated in the progression of kidney diseases. The purpose of this study was to investigate the potential role of urine cytology based on SurePath(™) combined with immunoenzyme staining using Wilms' tumour 1 (WT1) antibody as a podocyte marker in the discrimination of normality and non-renal urinary tract disease from kidney disease. METHODS: Sixty-six patients with kidney disease, 45 patients with lower urinary tract disease and 30 healthy volunteers were examined. Urine cytology slides were prepared using the SurePath method and immunoenzyme stained with WT1 antibody, and the number of WT1-positive cells was counted. RESULTS: In kidney disease, WT1-positive cells were found in 33 (50%) of 66 samples. No WT1-positive cells were found in 45 patients with lower urinary tract disease or in 30 healthy volunteers. The positive rates for WT1 varied with disease type, but not significantly: immunoglobulin A (IgA) nephropathy, (14/23); membranous glomerulonephritis, (4/10); Henoch-Schönlein purpura nephritis, (3/5); diabetic glomerulopathy, (5/5); minor glomerular abnormality/minimal change nephrotic syndrome (0/4). CONCLUSIONS: The results suggest that WT1 immunoenzyme staining of urine cytology can be used to detect some types of kidney disease.

A new cytokine-dependent monoblastic cell line with t(9;11)(p22;q23) differentiates to macrophages with macrophage colony-stimulating factor (M-CSF) and to osteoclast-like cells with M-CSF and interleukin-4.

Monocytes/macrophages exert a series of important functions in vivo. To facilitate detailed investigation of their functional capacity and the mechanism leading to their differentiation, several cell lines have been established from primary material. We present here a new human monoblastic cell line, designated UG3. UG3 cells are characterized by the following features. (1) UG3 cells harbor the t(9;11)(p22;q23) translocation that results in fusion of the MLL and the AF9 genes and produce the corresponding AF9-MLL and MLL-AF9 fusion transcripts. (2) UG3 cells rely on the presence of exogenous growth factors for viability and proliferation, such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), or macrophage colony-stimulating factor (M-CSF). (3) When cultured in the presence of G-CSF, UG3 cells differentiate along the granulocytic lineage, as evidenced by segmentation of nuclei and positive staining for neutrophilic alkaline phosphatase and peroxidase. (4) When cultured in the presence of GM-CSF or M-CSF, UG3 cells differentiate into mature macrophages while preserving surface expression of CD14 and CD68 and also start to release cytokines into cell-culture supernatants. Under these culture conditions, UG3 cells also take up acetylated LDL. (5) When cultured in the presence of M-CSF and IL-4, UG3 cells differentiate into osteoclast-like multinucleated giant cells capable of bone resorption and display tartrate-resistant acid phosphatase (TRAP) activity. UG3 cells thus provide features to qualify them as a useful model to further investigate the mechanism underlying these processes and also to further elucidate the functional role of mature monocytes/macrophages or osteoclasts.

Alterations of p53 and Rb genes in a novel human GM-CSF-dependent myeloid cell line (OHN-GM) established from therapy-related leukaemia.

A novel GM-CSF-dependent myeloid cell line, OHN-GM, was established from a patient who developed acute myelogenous leukaemia (AML) as a consequence of myelodysplastic syndrome (MDS). As the patient had previously received cytotoxic chemotherapy for Hodgkin's disease, the MDS and AML were probably related to such therapy. Sequential karyotypic analysis established a del(5q) as the initial cytogenetic abnormality. Additional alterations, including t(10;13)(q24;q14), had developed subsequently during disease progression. Southern blot analysis of OHN-GM cells suggested deletion of one allele of the IRF-1 gene, although no aberrant transcripts were detected. Fluorescence in situ hybridization analysis revealed the deletion of the Rb gene due to the t(10;13)(q24;q14) translocation, and Western blot analysis demonstrated the absence of Rb protein in OHN-GM cells. Finally, the OHN-GM cells exhibited two missense point mutations in highly conserved regions of the p53 gene. These observations suggest that a multistep process, involving alterations of Rb and p53 genes, may have contributed to the patient's disease development and progression. To our knowledge, OHN-GM is the first cell line derived from a therapy-related AML. These cells may aid the investigation of leukaemogenesis as well as the biology of secondary leukaemia.

GM-CSF- and IL-3-dependent CD34 expressing myeloid cell line (SAS-1) established from CD7 and CD34 expressing acute myeloblastic leukemic cells.

A novel factor-dependent human myeloid leukemia cell line (SAS-1) was established from a 69-year-old Japanese male suffering from CD7 and CD34 expressing acute myeloblastic leukemia (AML M2 in FAB classification). Morphological and cytochemical staining showed that SAS-1 cells were round with basophilic cytoplasm which is positive for peroxidase. Analysis of surface markers revealed that SAS-1 cells were myeloblasts derived from an immature progenitor origin, which express CD34. The consensus karyotype of the cell line was 41 XY 5q-, -7, 11p-, 12p+, -13, -14, -16, -17, -19, -22, with two markers. The proliferation of SAS-1 cells was dependent on the presence of either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), and GM-CSF- and IL-3-induced proliferation was dose dependent. Neither, stem cell factor (SCF) nor granulocyte colony-stimulating factor (G-CSF) alone supported the growth of SAS-1 cells, but supported their viability for more than 4 days and arrested them in the G0/G1 phase. SCF also enhanced GM-CSF- or IL-3-induced growth, but other cytokines did not have this synergistic effect. Clonogenic assays revealed that SAS-1 cells formed 36.0 +/- 5.7 or 41.5 +/- 0.7 colonies/1000 cells in the presence of GM-CSF or IL-3, respectively. SCF also increased the number of colonies formed by GM-CSF or IL-3 treatment, while SAS-1 cells did not form colonies in the presence of SCF alone. SAS-1 cells may prove to be a useful tool for studying the regulation of the cell cycle, myeloid proliferation, and differentiation.

Antisense src expression inhibits tyrosine phosphorylation of Shc and its association with Grb2 and Sos which leads to MAP kinase activation in U937 human leukemia cells.

We constructed a recombinant plasmid which expresses antisense src RNA after dexamethasone (Dexa) treatment, and transfected it into U937 human monoblastic leukemia cells (U937-ASRC). Induction of antisense src RNA expression diminished the amounts of c-Src and its protein tyrosine kinase (PTK) activity in U937-ASRC cells. The declines in c-Src and its PTK activity subsequently reduced the proliferation of U937-ASRC cells. To elucidate the growth signal transduction pathway downstream of c-Src, tyrosine phosphorylation of Shc was examined in U937-ASRC cells treated with Dexa. The decline in c-Src by induction of antisense src RNA expression decreased the level of tyrosine phosphorylation of Shc. Immunoprecipitated c-Src directly phosphorylated immunoprecipitated Shc on tyrosine residues in vitro. The amounts of Grb2 and Sos co-immunoprecipitated with Shc were decreased after Dexa treatment. However, the amount of Sos co-immunoprecipitated with Grb2 was apparently not affected by Dexa treatment. These results indicate that Grb2 and Sos constitutively associate with each other in U937 cells. Furthermore, the level of phosphorylation on tyrosine (204) essential for MAP kinase activation was decreased after Dexa treatment. Taken together with all these findings, it is suggested that c-Src directly phosphorylates Shc on tyrosine residues, which in turn binds to Grb2 constitutively associated with Sos to form a Shc-Grb2-Sos complex, and that the complex formation is coupled with MAP kinase activation mediated by Ras activation in U937 cells.

Synergistic cytotoxic effect of quercetin and heat treatment in a lymphoid cell line (OZ) with low HSP70 expression.

Heat shock protein 70 (HSP70) protects cells from various injurious stimuli and is important in cell growth and differentiation. However, its expression in leukemia cells has not been analyzed systematically. We, therefore, investigated the expression of HSP70 in various types of leukemia cells and hematopoietic cell lines. Immunoblot analysis revealed that HSP72/73 were expressed at low levels in the acute lymphocytic leukemia cells and lymphoid cell lines examined. However, heat (43 C for 2 h) preferentially augmented HSP72/73 expression in lymphoid cells. This induction was partially blocked by the treatment with quercetin (10 microM for 24 h). Finally, heat shock following quercetin treatment synergistically induced apoptosis in a lymphoid cell line (OZ). These observations suggest the possibility of selective purging of lymphocytic leukemia cells by combination with quercetin and heat.

Tumor vaccination with macrophage colony-stimulating factor-producing Lewis lung carcinoma in mice.

Expression of various cytokines by cytokine gene-transduced tumor cells has been shown to increase antitumor immunity of tumor-bearing hosts. In the present study, macrophage-colony stimulating factor (M-CSF) cDNA was retrovirally transfected into Lewis lung carcinoma cells (3LL) of C57BL/6 mouse origin, and the effects of M-CSF expression were studied by inoculating syngeneic C57BL/6 mice with M-CSF-expressing 3LL cells. The mice inoculated with the lowest M-CSF-producing 3LL clone showed significant prolongation of the survival compared with wild-type 3LL-Inoculated mice, and 70% or more of the mice inoculated with 3LL clones with higher M-CSF production rejected inoculation. Mice injected with radiation-inactivated M-CSF-expressing 3LL cells before or after Inoculation of wild-type 3LL cells showed prolonged survival compared with mice injected with radiated control 3LL cells before or after transplantation of wild-type cells. In vivo depletion of effector subpopulations by injection of antibodies against CD4+ T cells, CD8+ T cells, or natural killer (NK) cells suggested involvement of NK cells and CD4+ T cells in M-CSF-mediated antitumor cytotoxicity in M-CSF-producing 3LL cells-inoculated mice. Severe combined immunodeficiency (SCID) mice with defective T- and B-cell function showed prolonged survival duration after inoculation with M-CSF-expressing 3LL cells compared with those transplanted with control 3LL cells, and this effect of M-CSF expression by 3LL-cells in SCID mice was also abolished by in vivo depletion of NK cells by antibody injection. These findings together with the previous reports that M-CSF augments antibody-dependent and-independent antitumor cytotoxicity suggest that M-CSF induces tumor immunity in this cytokine-expressing tumor-transplantation model.

Thrombopoietin modulates platelet activation in vitro through protein-tyrosine phosphorylation.

To determine the roles of thrombopoietin (TPO) in platelet function in vitro, we examined the effects of TPO on platelet aggregation. Although several proteins in platelets were tyrosine-phosphorylated by TPO treatment, TPO alone was unable to induce platelet aggregation. However, the secondary wave of platelet aggregation induced by adenosine diphosphate (ADP) was enhanced by TPO in a dose-dependent manner. TPO in conjunction with ADP augmented tyrosine phosphorylation of platelet proteins, including tyrosine-phosphorylated proteins induced by TPO alone. Genistein inhibited protein-tyrosine phosphorylation in platelets induced by TPO with ADP and suppressed TPO-enhanced platelet aggregation. Moreover, tyrosine phosphorylation of MAP-kinases induced by TPO alone and TPO with ADP was consistent with TPO-enhanced platelet aggregation. These findings in the present study suggest that signal transduction involved in TPO-enhanced platelet aggregation is mediated in part by tyrosine-phosphorylated proteins, including MAP-kinases, in platelets through TPO-stimulated c-Mpl, TPO receptor.

Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, inhibits erythropoietin-induced erythroid differentiation of K562 cells.

To elucidate the role of phosphatidylinositol-3-kinase (Pl3K) in erythropoietin receptor (EPOR)-mediated signaling, we examined the effects of wortmannin (WT), a specific inhibitor of Pl3K on the proliferation of erythropoietin (EPO)-induced erythroid differentiation in K562 human erythroleukemia cells. Percentage of benzidine-positive cells synthesizing hemoglobin and level of glycophorin A expression in the cells were increased after EPO treatment. EPO-enhanced Pl3K activity was suppressed by WT treatment in a dose-dependent manner and constant inhibition of Pl3K by WT interfered with both hemoglobin synthesis and glycophorin A expression promoted by EPO. Wortmannin, however, did not inhibit hemin-induced erythroid differentiation. These findings in the present study suggest that Pl3K plays a crucial role in the transducing the erythroid differentiation signal through EPOR activated by EPO-binding ib K562 cells and that the signaling pathways involved in EPO-induced erythroid differentiation differ from those involved in hemin-induced differentiation.

Cell-to-cell interaction of cytokine-dependent myeloblastic line constitutively expressing membrane-bound stem cell factor abrogates cytokine dependency partially through granulocyte-macrophage colony-stimulating factor production.

Stem cell factor (SCF) is a cytokine for hematopoietic progenitor cells and plays an important role in megakaryocyte proliferation. The UT-7 cell line was established from a patient with megakaryoblastic leukemia, and its growth and survival are strictly dependent on interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (Epo), or IL-6. In this study, we showed that SCF also supported the growth of UT-7 in the absence of other cytokines and downregulated the cell surface c-kit receptors. Constitutive expression of SCF by introducing SCF expression vector made UT-7 grow factor-independently in liquid medium, but not in semisolid medium. This SCF-expressing factor-independent UT-7 (UT-7scf9) expressed the membrane bound form of SCF on their surface, but did not secrete detectable amounts of soluble SCF. UT-7scf9 formed aggregates as they grew in the absence of cytokines, and this aggregation was inhibited by adding soluble SCF into the medium. UT-7 cultured with SCF and UT-7scf9 cultured without cytokines expressed GM-CSF, and anti-GM-CSF neutralizing antibody partially inhibited their growth. These results suggest that SCF stimulated UT-7 proliferation partially through the autocrine-loop of GM-CSF, and UT-7scf9 expressed SCF mostly as a membrane-bound form, which transduces its growth signal through c-kit receptor as they aggregate by cell-to-cell interaction.

Effect of ubenimex on the proliferation and differentiation of U937 human histiocytic lymphoma cells.

We investigated the effect of ubenimex on the growth and differentiation of U937 cells, a histiocytic lymphoma cell line. Ubenimex is a dipeptide ((2S,3R)-3-amino-2-hydroxy-4-phenylbutyryl-L-leucine) and an inhibitor of aminopeptidase B produced by Streptomyces olivoreticuli. Ubenimex inhibited the proliferation of U937 cells in a dose-dependent manner. Ubenimex-treated U937 cells showed condensation of nuclear chromatin, increase of cytoplasmic vacuoles and more intense nonspecific esterase staining compared with untreated U937 cells. Expression of CD13 and CD68 detected by monoclonal antibodies My7 and EBM11, respectively, was enhanced by ubenimex, but the expression of CD4 detected by MT310 was significantly decreased. The effects of ubenimex on U937 cell growth inhibition and enhancement of monocytic cell surface marker expression on U937 cells were reversible when cultivated without ubenimex for more than 6 days. In addition, the bactericidal activity of U937 cells was increased by ubenimex treatment, and was further enhanced by treatment with macrophage colony-stimulating factor (M-CSF). Furthermore, ubenimex augmented the expression of M-CSF receptors by U937 cells and enhanced the tyrosine kinase activity of cellular pp60c-src. These findings indicated that ubenimex inhibited the proliferation of U937 cells and induced morphological, cytochemical and functional differentiation into monocyte/macrophages.

Low-dose continuous subcutaneous infusion of granulocyte colony-stimulating factor for chemotherapy-induced neutropenia in acute myelogenous leukemia and its pharmacokinetics.

Granulocyte colony-stimulating factor (G-CSF) shortens the duration of chemotherapy-induced granulocytopenia in acute leukemia. G-CSF is administered by 30-min intravenous infusion at a dose of 200 micrograms/m2/day or 5 micrograms/kg/day in most studies. In this study, the efficacy of a reduced dose (33 micrograms/m2/day) of continuous subcutaneous infusion of G-CSF was compared with the effects achieved by 30-min intravenous infusion of the standard dose (200 micrograms/m2/day) in neutropenia after identical chemotherapy in seven patients with acute myelogenous leukemia who were in remission. The duration of granulocytopenia (< 0.5 x 10(9)/l), thrombocytopenia (< 50 x 10(9)/l); G-CSF administration and fever (> 38 degrees C) were 10.1 +/- 5.0 days, 16.5 +/- 9.3 days, 16.6 +/- 7.4 days and 3.1 +/- 5.4 days for 33 micrograms/m2/day continuous subcutaneous infusion, and 10.7 +/- 6.8 days, 16.7 +/- 9.9 days, 16.1 +/- 7.6 days and 2.0 +/- 2.5 days for 30-min intravenous infusion of the standard dose of G-CSF. In each parameter studied, there was no statistical difference between the two methods of G-CSF administration by paired t-test. The costs for G-CSF could be substantially reduced. In most patients, plasma G-CSF concentration rose to the highest level of 1.8-3.7 ng/ml 48-72 h after starting 33 micrograms/m2/day continuous subcutaneous infusion, and gradually decreased as the peripheral granulocyte count recovered, suggesting binding of G-CSF molecules to the specific receptors on the cells of granulocytic lineage.

PML-RAR alpha fusion transcripts by RNA PCR in acute promyelocytic leukemia in remission and its correlation with clinical outcome.

Molecularly defined specific chromosomal translocation in leukemia allows detection of minimal residual leukemia cells by the reverse transcription-polymerase chain reaction (RT-PCR). However, the positivity of the specific fusion transcripts in chronic myelogenous leukemia and acute myelogenous leukemia with t(8;21) is reportedly not directly correlated with the predictability of relapse. We analyzed seven patients with acute promyelocytic leukemia (APL) in hematological remission for PML-retinoic acid receptor alpha (PML-RAR alpha) fusion transcripts by RT-PCR with the sensitivity level of one APL cell in 10(5) bone marrow mononuclear cells. Two of the four patients with chemotherapy-induced remission had detectable PML-RAR alpha only before treatment. In the other two patients with chemotherapy-induced remission, the PML-RAR alpha was detectable when their remission was first confirmed and became negative after consolidation chemotherapy. Two patients were resistant to chemotherapy and achieved remission by all-trans-retinoic acid; PML-RAR alpha was detectable in them for a few months during consolidation chemotherapy. Two patients whose PML-RAR alpha had become continuously positive had relapse 2 and 8 months later, but the other five patients with continuously negative or only transiently positive PML-RAR alpha remained in remission during follow-up for 11 to 35 months. These findings suggest the relevance of detectable PML-RAR alpha by RT-PCR to the predictability of relapse in acute promyelocytic leukemia.

Effects of the antisense myb expression on hemin- and erythropoietin-induced erythroid differentiation of K562 cells.

In order to elucidate the role of c-myb gene in erythroid differentiation of K562 cell induced by hemin (Hm) and erythropoietin (Epo), we constructed recombinant plasmid that could produce antisense myb RNA after induction with dexamethasone. During treatment with Hm, K562 cells constitutively expressed c-myb mRNA, and 50% of them began to synthesize hemoglobin (Hb). Expression of antisense myb RNA reduced the amount of c-myb mRNA, and the percentage of Hb-synthesizing cells was decreased to 20%. In the presence of Epo, c-myb mRNA declined and 20% of K562 cells synthesized Hb regardless of antisense myb RNA expression. It is suggested that constitutive expression of c-myb mRNA is necessary for Hm-induced differentiation, and that a decrease in the amount of c-myb mRNA induced by antisense myb RNA expression suppresses Hm-induced differentiation. The amount of c-myb mRNA in K562 cells was reduced during the differentiation induced by Epo. Expression of GATA-1 mRNA was almost constant during Hm-induced differentiation, but increased during Epo treatment. It is supposed that the mechanism of Hm-induced differentiation is distinguished from that of Epo-induced differentiation in K562 cells.

Antisense src expression inhibits proliferation and erythropoietin-induced erythroid differentiation of K562 human leukemia cells.

We constructed a recombinant plasmid which expresses antisense src RNA in human cells and used it as a tool for investigating the role of pp60c-src in proliferation and differentiation of K562 human leukemia cells. In erythropoietin (EPO)-responsive cells, EPO induces rapid tyrosine phosphorylation of several cellular proteins including EPO receptor (EPOR) although EPOR has no tyrosine kinase domain. Here we show that antisense src RNA expression suppresses pp60c-src synthesis in the recombinant plasmid-transfected K562 cells, reduces the proliferation and inhibits hemoglobin synthesis and glycophorin A expression promoted by EPO in K562 cells. These findings suggest that pp60c-src plays crucial roles in the proliferation and EPO-induced erythroid differentiation of K562 cells.

Antisense src expression inhibits U937 human leukemia cell proliferation in conjunction with reduction of c-myb expression.

To elucidate the role of pp60c-src in U937 human monoblastoid leukemia cell proliferation, recombinant plasmids containing the src gene or myb gene, which could produce antisense src or antisense myb RNA after dexamethasone treatment, were constructed and transfected into U937 cells (U937-ASRC, U937-AMYB). pp60c-src synthesis in U937-ASRC was diminished by the third day after induction of antisense src RNA and the cell proliferation was reduced, furthermore, the amount of p75c-myb was significantly decreased by the third day. p75c-myb synthesis in U937-AMYB was diminished by the second day after induction of antisense myb RNA and the cell growth was significantly inhibited but the amount of pp60c-src in U937-AMYB was not reduced. These results suggest that a decrease in the amount of pp60c-src leads to an inhibition of p75c-myb expression and subsequent reduction in the U937 cell proliferation.