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.

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.

Behavior of activities of thymidine metabolizing enzymes in human leukemia-lymphoma cells.

The behavior of the activities of thymidine metabolizing enzymes, dihydrothymine dehydrogenase (EC 1.3.1.2) and thymidine phosphorylase (EC 2.4.2.4) for thymidine degradation, thymidine kinase (EC 2.7.1.75) and thymidylate synthase (EC 2.1.1.45) for DNA synthesis, was elucidated in cytosolic extracts from normal human lymphocytes and 13 human leukemia-lymphoma cell lines. In the normal human lymphocytes, the activities of dihydrothymine dehydrogenase, thymidine phosphorylase, thymidine kinase, and thymidylate synthase were 6.88, 796, 0.30, and 0.29 nmol/h/mg protein, respectively. In leukemia-lymphoma cell lines, the activities of synthetic enzymes, thymidine kinase, and thymidylate synthase, increased two- to 79-fold and 22- to 407-fold of the normal lymphocyte values. In contrast, the activities of the catabolic enzymes, dihydrothymine dehydrogenase and thymidine phosphorylase, decreased to 5-42% and 3-38% of the values of normal lymphocytes. As a result, the ratio of activities of thymidine kinase/dihydrothymine dehydrogenase was elevated by 7- to 1170-fold, respectively. Thus, reciprocal behavior in the activities of the opposing enzymes in thymidine metabolism was observed in human leukemia-lymphoma cells. Polyclonal and monoclonal antibodies against dihydrothymine dehydrogenase were prepared and studies on immunotitration of this enzyme with these antibodies showed that the enzyme protein amount in Jurkat leukemic cells was 36% of that of normal lymphocytes. This was in good agreement with the decrease in the activity of the enzyme to 32%, indicating that the decrease in activity in the leukemic cells was due to the decline in the amount of enzyme protein. The metabolic imbalances in thymidine utilization appear to be characteristic of human leukemia-lymphoma cells. These observations should confer selective advantages to the lymphoproliferating cells and mark out the catabolic, as well as the synthetic, enzymes as important targets in the design of chemotherapy.

Selective up-regulation of type II inosine 5'-monophosphate dehydrogenase messenger RNA expression in human leukemias.

The discovery of isozymes (types I and II) of IMP dehydrogenase (IMPDH; EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, has attracted attention as a possible novel approach to cancer diagnosis and selective tumor cell chemotherapy. To elucidate differences in expression and regulation of the two IMPDH isozymes, we examined the steady-state levels of these mRNAs in various types of leukemic cells from patients. Northern blot analysis revealed that type II IMPDH was more active transcriptionally (1.5- to 5.1-fold) in all the leukemic cells examined than in normal lymphocytes, whereas type I expression was similar. The increased expression of type II mRNA in leukemic cells was closely linked with the increase in total IMPDH activity (r = 0.92). When leukemic cells from a patient with chronic granulocytic leukemia in blast crisis were separated into blast-rich and mature leukocyte-rich fractions, the expression of type II mRNA correlated positively with the population of immature leukemic cells, whereas type I expression was unchanged. Treatment of leukemic blasts with 12-O-tetradecanoyl-phorbol-13-acetate for 5 days resulted in a 90% decrease in the expression of type II mRNA with macrophage-like differentiation, while the expression of type I mRNA was relatively stable. These observations suggest that expression of type II IMPDH is stringently linked with immature characteristics of leukemic cells; thus, it should be a selective target for antileukemic chemotherapy.

Decreased accumulation as a mechanism of resistance to cis-diamminedichloroplatinum(II) in human non-small cell lung cancer cell lines: relation to DNA damage and repair.

The mechanism of resistance to cis-diamminedichloroplatinum(II) (CDDP) is still controversial, although several kinds of processes have been proposed. For elucidation of the mechanism of CDDP resistance in CDDP-resistant human non-small cell lung cancer cells (PC-9/0.5, 7.1-fold resistant), we examined the formation of DNA interstrand cross-links (ICL), one kind of DNA damage induced by CDDP, its repair, and intracellular accumulation of CDDP. We measured the frequency of CDDP-induced ICL by means of the alkaline elution technique and the amount of intracellular platinum for intracellular accumulation of CDDP by means of atomic absorption spectrophotometry in PC-9 (parental cell) and PC-9/0.5 cells. Formation of ICL in PC-9 cells exposed to 5 micrograms of CDDP per ml for 6 h was 5.85 times that in PC-9/0.5 cells. On the other hand, the ability to repair CDDP-induced ICL was identical in both cell lines. Intracellular accumulation studies revealed that PC-9 retained 5.07 times as much platinum as that in PC-9/0.5 after 3-h exposure to CDDP. It was conjectured that the decrease in the intracellular accumulation of CDDP might be the main cause of CDDP resistance in PC-9 cells, since the decreased accumulation was paralleled by the decreased level of ICL.

Reversal of enzymic phenotype of thymidine metabolism in induced differentiation of U-937 cells.

Exposure of U-937 cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in specific alterations in thymidine metabolism. Within 24 h after treatment with 1.62 x 10(-9) M TPA, the reciprocal alteration in the activities of opposing enzymes of thymidine metabolism observed during normal cell culture growth was reversed. In TPA-treated cells, the activities of anabolic enzymes thymidine kinase (EC 2.7.1.75)and thymidylate synthase (EC 2.1.1.45) declined with time linearly to 20 and 16% of those of untreated cells by 72 h. Incorporation of [3H]thymidine and [3H]deoxyuridine into acid-insoluble fractions also decreased in parallel with the decline in enzyme activities. In contrast, the activities of catabolic enzymes thymidine phosphorylase (EC 2.4.2.4) and dihydrothymine dehydrogenase (EC 1.3.1.2) increased. The rise in thymidine phosphorylase activity peaked at 48 h with 406% elevation over the control. The activity of dihydrothymine dehydrogenase was not altered for the first 24 h, but it increased up to 338% by 96 h. Immunotitration of dihydrothymine dehydrogenase with monoclonal antibody against this enzyme showed that the rise in activity in the differentiated cells was due to the increase in the amount of enzyme protein. No significant difference was observed in the Km values for the substrate of each enzyme between untreated and TPA-treated cells. These metabolic alterations during induced differentiation were in line with the changes in cell morphology and accompanied by an accumulation of the cells in G1 at the expense of S phase. These observations indicate that induced differentiation of U-937 cells results in a reversal of the enzymic phenotype of thymidine metabolism and suggest that emergence of thymidine metabolic imbalance may serve as an early marker of differentiation of these cells.

Genomic rearrangement of the c-myc proto-oncogene in non-AIDS-related lymphoma in Japan.

Southern blot analysis was employed to analyze the structural alterations of the c-myc oncogene in genomic DNA derived from tumor specimens of 35 adults with pathologically classified and immunophenotyped non-AIDS-related, non-Hodgkin's lymphoma in Japan. In this study, seven cases (20%), including one peripheral T-cell lymphoma and six B-cell lymphomas of various histological types, were demonstrated to have additional c-myc fragments. An interesting feature is that c-myc rearrangements were found in three out of eight primary gastrointestinal lymphomas. Analyses with several restriction enzymes revealed that the breakpoints in these cases were clustered in a region spanning the first exon, first intron and nearby 5'-flanking sequences of the c-myc gene, suggesting that the alteration of this region may represent an important molecular event in activating the oncogenic potential of the c-myc gene.

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.

Reverse transcription-polymerase chain reaction for PML-RAR alpha fusion transcripts in acute promyelocytic leukemia and its application to minimal residual leukemia detection.

Chromosome translocation t(15;17) specifically found in acute promyelocytic leukemia (APL) results in cleavage in the introns of PML gene on chromosome 15 and in the intron of the retinoic acid receptor alpha (RAR alpha) gene on chromosome 17, creation and expression of PML-RAR alpha and RAR alpha-PML fusion genes. Reverse transcription-polymerase chain reaction (RT-PCR) was applied to detect the PML-RAR alpha fusion transcripts rapidly in APL patients. The fusion transcripts could be detected in all of the 10 APL patients studied. Of the two breakpoints in the PML gene so far reported, seven APL patients had the fusion transcript compatible with the downstream (3') breakpoint, and the other three APL patients were considered to have the upstream (5') breakpoint. RT-PCR could detect the fusion transcripts from as little as 50 pg bone marrow RNA, and from as little as 0.5 pg bone marrow RNA with the nested PCR. This method was applied to detect minimal residual leukemia cells in an APL patient who had undergone allogeneic bone marrow transplantation, in whom the RT-PCR could not detect the PML-RAR alpha fusion transcripts at several post-transplant time points. This system could be useful to detect minimal residual leukemia cells and accordingly modify the treatment strategy, as well as to make a quick diagnosis with a small amount of clinical sample.

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.

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.

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.

Physiological role of growth hormone (GH)-releasing factor and somatostatin in the dynamics of GH secretion in adult male rat.

To investigate the physiological role of GRF and somatostatin (SRIF) in GH secretion in adult male rats, we prepared in vitro models using the perifusion system of cultured rat anterior pituitary cells exposed to various combinations of human GRF-(1-44)NH2 (hGRF) and SRIF. We studied the following three models on GRF secretion: 1) pulsatile GRF secreted at 1-h intervals, 2) pulsatile GRF secreted at 3-h intervals, and 3) GRF continuously secreted. When 5-min pulses of 20 nM GRF were delivered at 1-h intervals, the responses to GRF gradually declined. The addition of continuous 20 nM SRIF with short pauses prevented this attenuated response and produced high peaks of GH at 3-h intervals. When 5-min pulses of 20 nM GRF were delivered at 3-h intervals, three high peaks of GH were observed regardless of the addition of SRIF. Pretreatment with GRF pulses enhanced the peaks of GH during SRIF pauses. When 20 nM GRF was continuously delivered, a rapid attenuated response to GRF was observed. Although the addition of continuous 20 nM SRIF with short pauses produced three small peaks of GH, these results were caused by the post-SRIF rebound release. These observations suggest that preexposure to prolonged SRIF can prevent an attenuated response to repeated GRF pulses, and that pretreatment with GRF pulses enhances post-SRIF rebound release.

Isolation of a cDNA clone encoding DNA-binding protein (TAXREB107) that binds specifically to domain C of the tax-responsive enhancer element in the long terminal repeat of human T-cell leukemia virus type I.

Five cDNA clones for TAXREB proteins that bind to the tax-responsive enhancer element of human T-cell leukemia virus type I (HTLV-I) were isolated from a Jurkat cell cDNA library. The beta-galactosidase fusion proteins of three of these clones specifically recognized the domain C within the enhancer. One of the three cDNAs, encoding TAXREB107, contained an open reading frame with 288 amino acid residues. RNA blot analysis showed that the level of mRNA for TAXREB107 increased transiently in Jurkat cells on treatment with TPA. Immunoblot analysis showed that polyclonal antibody against TAXREB107 specifically recognized a 34-kD protein in Jurkat cells. TAXREB107 may participate in tax-mediated trans-activation of transcription.

Induction of erythroid differentiation of K562 cells by 4-carbamoylimidazolium 5-olate (SM-108).

The effects of 4-carbamoylimidazolium 5-olate (SM-108), an antipurine compound, on a human leukemia cell line, K562, were studied. Treatment with SM-108 induced erythroid differentiation of K562 cells. During a 6-day culture with 100 microM SM-108, the cell number decreased to 37% of the control number, 77% of the cells became benzidine-positive, and the hemoglobin content increased from 2.1 +/- 0.2 to 10.6 +/- 1.3 pg/cell. Cell differentiation was associated with reduction of IMP dehydrogenase activity and intracellular GTP content to 25 and 36%, respectively, of the control values within 1.5 hr. The differentiation and decrease in the GTP pool induced by SM-108 were blocked by the presence of 25 microM guanine or guanosine. SM-108 also induced erythroid differentiation of K562 subline cells transfected with pMSG (K562/pMSG), which have an additional salvage pathway for GMP production from xanthine. The addition of 100 microM xanthine prevented erythroid differentiation of this subline and restored the GTP pool. These findings suggest that the induction of erythroid differentiation of K562 cells by SM-108 may be due to an early decrease in IMP dehydrogenase activity and a subsequent decrease in GTP content in the cells. Thus, purine metabolism may have an important role in SM-108-induced differentiation.

1,25(OH)2-vitamin D3 mediated changes in mRNA for c-myc and 1,25(OH)2D3 receptor in HL-60 cells and related subclones.

HL-60 cells have been shown to differentiate into monocyte-like cells as a consequence of the interaction of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) with a specific intracellular receptor (VDR). Of c-myc it has been reported that it plays a role in cell proliferation and differentiation. We have investigated the expression of VDR and c-myc mRNA of three subclones of HL-60 cells (N, R and MR) after exposure to 1,25(OH)2D3. The N cell is a normal clone in which 1,25(OH)2D3 inhibited cell proliferation and also induced cell differentiation. The R cell is a resistant clone whose proliferation and differentiation are not affected by 1,25(OH)2D3. The MR cell is a mixed response type clone whose proliferation was not inhibited by 1,25(OH)2D3, while its differentiation was actually induced by 1,25(OH)2D3. The VDR mRNA expression of the N and MR cells reached a peak at 2 h (2.3- and 2.6-fold induction, respectively) and returned to the control level after 24 h treatment of 1,25(OH)2D3. The c-myc mRNA expression was significantly inhibited in the 1,25(OH)2D3-induced N cells, but not in the MR cells. In contrast, 1,25(OH)2D3 did not induce any changes of the VDR and c-myc mRNA levels in the R cells. In separate experiments, the level of the VDR in the three clones was determined via Scatchard analysis; the VDR of the N, MR and R cells were found to be 4500/cell, 3570/cell and less than 600/cell, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies raised against K562 cells reacted with human haematopoietic pluripotent stem cells.

We describe the properties of three monoclonal antibodies (McAbs) (21H73, 37G7 and 49C12) against K562 cell surface antigens correlated with differentiation induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA). Each of the McAbs immunoprecipitated K562 cell surface antigen with molecular weight (MW) of approximately 51 kD, 82 kD or 92 kD, respectively. The antigens detected by McAbs 21H73 and 37G7 were not immunoprecipitated from K562 cells differentiated into monocyte-macrophages by TPA (K562-TPA). On the other hand, 49C12 immunoprecipitated an antigen with MW of 92 kD from K562-TPA cells, but not from undifferentiated K562 cells. To examine the distribution of these antigens among human haematopoietic stem cells, bone marrow cells were separated by the panning method using these McAbs and subjected to colony-forming assays. The McAb 21H73 reacted with CFU-mix and BFU-E but with neither CFU-E nor CFU-GM. CFU-mix and BFU-E were enriched approximately 6.2-14.7-fold and 2-fold by the panning procedure using 21H73, respectively. On the other hand, 37G7 reacted only with BFU-E, and 49C12 reacted with CFU-GM but not with any other haematopoietic progenitor cells. We also examined the reactivity of these McAbs with leukaemia cells freshly isolated from 26 patients. The antigen defined by 21H73 was not expressed on any leukaemia cells from patients except for cells from an acute lymphocytic leukaemia (ALL, L3) and a CML in blastic crisis. The McAb 37G7 reacted with several types of leukaemia cells. The antigen defined by 49C12 was expressed on almost all leukaemia cells isolated from patients. These results suggest that 21H73 allows purification and enrichment of normal haematopoietic pluripotent stem cells from both normal and leukaemia patients' bone marrow specimens, especially following the step to remove leukaemia cells and haematopoietic progenitor cells other than CFU-mix by using 37G7 and/or 49C12.

p75c-myb expression in leukemia-lymphoma cells correlated with proliferation and differentiation.

Monoclonal antibodies (McAbs) against a part of v-myb gene product were prepared for the detection of human c-myb gene product (p75c-myb). Western blotting analyses with these McAbs were performed on human leukemia-lymphoma cells. All T-cell lines were positive in p75c-myb expression. B-cell lines were variable, myeloid and erythroid cells were positive although the amount of expressed p75c-myb was less than the T-cell lines. Cells isolated from patients were positive in expression except for cells from acute myeloblastic leukemia with maturation (AML M2), acute hypergranular promyelocytic leukemia (AML M3) and erythroleukemia (AML M6) developed from myelodysplastic syndromes. Differences in p75c-myb expression seemed to depend upon the differentiation stage and distinctive lineage from which each cell line had been established. The p75c-myb expression in HL60 (acute promyelocytic leukemia cell line) showed remarkably high at logarithmic growth. When examined with HL60, p75c-myb expression significantly decreased during the differentiation induced by 12-O-tetradecanoylphorbol-13-acetate or retinoic acid. These results suggest that p75c-myb expression plays a crucial role in hematopoietic cell proliferation and differentiation and that multiple mechanisms including aberrant expression of p75c-myb is involved in leukemogenesis.

Usefulness of a small scale indirect 125I-labelled protein A binding assay for detection of monoclonal antibodies against hematopoietic cell surface antigens.

We describe a small-scale solid-phase indirect 125iodine protein A binding assay (IPA) and discuss its usefulness for detection of hematopoietic cell surface antigens and for screening hybridoma clones producing monoclonal antibodies (McAbs) against K562 cell surface antigens correlated with differentiation induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA). This assay was performed in polystyrene Terasaki microtest plates instead of 96-well microtiter plates, using hematopoietic cells attached to the wells by air drying. This method facilitates washing procedures and reduces the radioactive waste. The specificity of this IPA method is as high as those of RIAs and ELISAs generally used. Titration curves of a McAb, My7, specific for CD13 on HL60 and TPA-treated HL60 (HL60-TPA) cells, were linear between the 10(-1) and 10(-4) dilutions. A difference in the CD13 expression between HL60 and HL60-TPA cells could be detected by both IPA and flow cytometry using My7 at 10(-1)-10(-3) dilutions. At the 10(-3) dilution, however, CD13 expressed on HL60 cells was detected by the IPA method but not by flow cytometry. These results show that the sensitivity of IPA is superior to that of flow cytometry. By screening hybridoma clones with this IPA method, we succeeded in producing three interesting and useful McAbs (21H73, 37G7 and 49C12) against K562 cell surface antigens whose expression was altered after the differentiation induced by TPA. We also demonstrated that the IPA method is suitable for cell surface marker analysis of leukemia cells freshly isolated from patients.

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.