Possible prognostic value of nucleolar morphology in pathologic cells of B-chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) represents a heterogeneous disease with a very variable outcome. The reliable prognosis of this disease at the time of initial diagnosis is difficult to predict. The purpose of this preliminary study was to utilize the nucleolar morphology and to investigate the incidence of main nucleolar types in leukemic lymphocytes in B-CLL patients to assess their possible predictive value for the disease outcome, in correlation with immunophenotype parameters. The evaluation of nucleolar morphology of pathologic lymphocytes was performed at diagnosis and during the course of disease. Median follow up period of patients was 16.4 months (range from 2 to 32 months) from diagnosis. The nucleoli were visualized by a simple cytochemical demonstration of RNA and the proportion of main nucleolar types in pathologic lymphocyte population infiltrating bone marrow of 84 patients suffering from B-CLL was analyzed. The presence of ring shaped and compact nucleoli in leukemic lymphocytes divided patients into two subgroups with different outcome of the disease. Malignant lymphocytes of the majority of patients (Group 1, 71 patients, 84.5%) mostly contained ring shaped nucleoli. These patients were in stable phase and did not require any treatment during the follow up. The population of leukemic cells of a small group of B-CLL patients (Group 2, 13 patients, 15.4%) was characterized by the presence of various proportions of pathologic lymphocytes with one large compact nucleolus.Different response to the therapy discriminated the B-CLL patients whose leukemic lymphocytes revealed evident compact nucleoli at presentation, to next two subsets. Four of these patients (Group 2, 4/13, 31%) appeared to be resistant to chemotherapy, others (9/13, 69%) showed response to therapy, though the response time was variable. Leukemic cells with compact nucleolus morphologically resembled prolymphocytes, but hematologically and immunophenotypically did not fulfill the diagnostic criteria for prolymphocyte population. None of our B-CLL patients had the signs of transformation to prolymphocytic or other type of B cell neoplasms during the follow up. Our results indicate the possibility of relationship between the presence of malignant lymphocytes with compact nucleoli and unfavorable outcome in patients with B-CLL. The simplicity and utility of the nucleolar test as a possible prognostic parameter may help to identify the subset of patients with early B-CLL disease that will run a more progressive course.

Myeloid enzymes profile related to the immunophenotypic characteristics of blast cells from patients with acute myeloid leukemia (AML) at diagnosis.

The purpose of this study was to assess the possible relationship between the cytochemical enzyme profile and immunophenotypic characteristics of distinct acute myeloid leukemia (AML) subtypes in discrete stages of leukemic cells maturation. As the proportion of leukemic blast cells is critical for exact cytochemical analysis, study was restricted to the evaluation of 48 adult and pediatric patients with newly diagnosed AMLs with 80% or more blasts in analyzed samples. The cytochemical investigation of myeloperoxidase (MPO), Sudan black B (SBB), chloroacetate esterase (CAE), alpha-naphthyl butyrate esterase (ANBE), alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AP) in peripheral blood and/or bone marrow was performed. The immunophenotype was examined for the maturation dependent myeloid antigens CD13, CD33, CD11b, CD14, CD15, CD65, CD36, cytoplasmic MPO, non-lineage associated CD34 and HLA-DR antigens, lymphoid- associated antigens CD7, CD4, CD38 as well as natural killer cell associated marker CD56. Flow cytometry by double marker staining and visualization of pathologic cells in dot plots reflected immunophenotypic aberrancy and degree of cell maturation. The patients were classified into AML subtypes M0- M2, M3, M4 and M5 according to the main morphological, cytochemical and immunophenotypical features. The variable combinations of MPO, SBB, CAE and ANBE were identified in relation to immunophenotype. The cytochemical profile of blasts was in concordance with immunophenotype, particularly in more differentiated AML subtypes, M3, M4 and M5. The findings of myeloid antigens expression and cytochemical features in poorly differentiated AML subtypes showed no practical relevance of cytochemical analysis. Notwithstanding that the cytochemical analysis of AML subtypes not sufficiently identifies the distinct aberrancies in heterogeneous leukemic blast cell populations, evaluation of the cytochemical profile in connection with immunophenotyping may help to classify the AML patients to relevant subtypes with more accuracy.

Argyrophilic nucleolar organizer regions (AgNORs) in relation to p53 and bcl-2 protein expression in leukemia patients.

The aim of this study was to assess the possible relationship between the silver stained nucleolar organizer regions (AgNOR) and immunocytochemically detected p53 and bcl-2 proteins in ALL, AML, B-CLL and CML patients (adults and children) at the initial presentation. AgNORs are loops of DNA, correlated with proliferative potential of cells. Alteration in p53 and bcl-2 proteins expression may characterize the malignant potential of leukemic cells. The patients were subdivided according to the p53 positivity and negativity. The frequency of p53-positive patients was relatively low in T-ALL (29%) and B-CLL (16%). B-ALL, AML and CML patients revealed higher frequency of p53 protein (46%, 47% and 88%, respectively). The overall frequency of positive cytoplasmic staining for bcl-2 protein was demonstrated in the majority of patients. No significant differences in the percentage of p53-positive cells among leukemia subtypes were seen. The proportion of bcl-2 protein positive cells did not differ significantly among various leukemia subtypes, except for significant differences between p53-positive and p53-negative peripheral blood (p=0.0073) and bone marrow (p=0.0175) cells of B-CLL patients. The samples from healthy subjects used as controls exhibited relatively low numbers of AgNOR dots in both, peripheral blood and bone marrow cells. Highly significant differences in AgNOR quantities between healthy donors and p53 protein positive peripheral blood as well as bone marrow cells of distinct leukemia subtypes (except for bone marrow cells in B-CLL patients, p=0.1727) were observed. Significant differences in AgNOR count between p53 protein positive and p53 protein negative samples of peripheral blood cells of B-ALL (p=0.0099) as well as B-CLL (p=0.0117) cases were found. No significant differences (except for B-CLL, p=0.0558) were encountered in bone marrow cells. P53 protein positivity or negativity did not influence the amount of AgNOR proteins in cells of our T-ALL and AML cases. Mutual comparing the number of AgNOR dots among different leukemias showed that for both peripheral blood and bone marrow cells the differences between ALL and AML (p=0.0383 and p=0.0033, respectively) as well as for peripheral blood of AML and CML (p=0.0302) were statistically significant. The bcl-2 protein positivity did not affect significantly the AgNOR distribution either in p53 protein positive or p53-negative cases of our leukemia patients. However, an association between the lowest AgNOR quantity and highest bcl-2 protein expression in p53-negative B-CLL patients was seen for both peripheral blood and bone marrow cells. The correlation between relatively high AgNOR numbers and relatively increased percentage of bcl-2 protein in the p53-positive cases of CML patients was found in some cases. Regarding the age and sex, the AgNOR distribution in p53-positive and p53-negative leukemia cases in children and adults showed neither relationship nor dependence. The WBC count differed evidently among distinct leukemia subtypes, with enormous heterogeneity in range as well. Larger studies are needed in order to consolidate these preliminary results and characterize the possible prognostic value of AgNOR in association with p53 and bcl-2 proteins expression.

Immunophenotyping of leukocytes in peripheral blood of BALB/c mice infected with mouse herpesvirus isolate 72.

We characterized leukocytes in peripheral blood of BALB/c mice infected with mouse herpesvirus isolate 72 (MHV-72) representing an isolate of mouse herpesvirus strain 68 (MHV-68, species Murid herpesvirus 4, genus Rhadinovirus, subfamily Gammaherpesvirinae, family Herpesviridae) (van Regenmortel et al., 2000). In acute infection (up to day 30 post infection (p.i.)) the number of CD8+ T cells increased, reaching a maximum at day 11 p.i. This increase correlated with that of CD4+ T, activated CD 19+ B and natural killer (NK) cells. At day 30 p.i. the numbers of CD4+, CD8+, CD14+ and CD19+ cells decreased to normal values. A similar increase in the number of these cells was observed at day 730 p. i. In the course of persistent infection (after day 30 p.i.) some of the mice developed a leukemia-like syndrome characterized by an increase in the number of leukocytes and appearance of atypical, blastic immature forms of leukocytes. The latter forms of leukocytes were characteristic by an increased amount of argyrophilic proteins. These results show further similarities between MHV-72 (another isolate of MHV-68) and EBV infections and justify the use of MHV-68 or MHV-72 as an appropriate mouse model for the study of EBV infection of humans.

Immunocytochemical detection of bcl-2 and p53 proteins in B-chronic lymphocytic leukemia patients.

B-cell chronic lymphocytic leukemia (B-CLL) is a disease with variable course and prognosis. It may be important to predict the possible risk of disease progression in individual patients. We have investigated by immunocytochemistry the bcl-2 and p53 protein expression in 53 B-CLL patients at the time of initial diagnosis. All B-CLL cases were bcl-2 protein positive. The relatively high frequency of p53 protein immunoreactivity was observed (17 of 53 cases; 32%). The percentage of bcl-2 and p53 protein positive cells remarkably varied in individual patients. The heterogeneity in the percentage of bcl-2 as well as p53 positive cells showed to be important in the analysis of mutual relation of these proteins. Noteworthy results were obtained when the group of p53 positive B-CLL patients was analyzed according to the percentage of p53 positive cells (less than 20% and more than 20%, respectively). An inverse relationship between a higher accumulation of p53 and repressed bcl-2 expression and vice versa was observed. The male patients (female patients were not assessed because of the limited number of p53 positive cases) with less than 20% p53 immunoreactive cells revealed a high percentage of bcl-2 protein (p=0.0008). The higher incidence (over 20%) p53 positive cells correlated with lowered percentage of bcl-2 positive cells (p=0.0368). When the patients were subdivided according to p53 positivity and negativity, the majority of p53 positive cases were males (82%), with significantly higher WBC count (p=0.0362). No significant effect of higher or lower WBC counts on bcl-2 and p53 expression was observed. However, the expression of bcl-2 protein was significantly higher in female patients under 50 years (p=0.0012). Regarding the patients of age 50, a significant difference in WBC count was shown in males (p=0.0590). The peripheral blood lymphocytes isolated from healthy subjects used as controls, exhibited undetectable, to low proportion of bcl-2 and p53 positive cells. Comparing the percentage of bcl-2 as well as p53 positive cells in controls and those of B-CLL patients, the significant difference for both proteins was observed (p=0.0009 and p=0.0001, respectively). The results of this study indicate that the overexpression of p53 protein may contribute to the downregulation of bcl-2 in a subgroup of our B-CLL patients. Considering the small numbers of tested p53 positive cases, it would be necessary to confirm our findings in a larger cohort of patients with longer follow up. Thus it would be possible to confirm our expectation of a possible value of the simultaneous aberrant expression of bcl-2 and p53 as useful predictors of future aggressive behavior of B-CLL.

Expression of p53 and bcl-2 proteins in acute leukemias: an immunocytochemical study.

We have analyzed by immunocytochemistry the p53 and Bcl-2 proteins expression in 49 patients with B-ALL, T-ALL and AML at the time of initial diagnosis. The diagnosis was based on morphologic and cytochemical criteria and on immunophenotyping. To demonstrate the p53 protein expression, p53 specific mouse antihuman immunoreagent clone DO-1 that recognizes both wild and mutated p53 protein was used. To detect Bcl-2 a monoclonal antibody that recognizes the 26-kD Bcl-2 protein was applied. For evaluation of both proteins a sensitive Immunotech detection kit based on peroxidase labeled streptavidin biotin reagent was utilized. The patients were divided according to the presence or absence of both, nuclear p53 and cytoplasmic Bcl-2 proteins. A relative low frequency of p53 protein expression in B- and T-lineage acute lymphoblastic leukemia has been shown at diagnosis. In AML cases, the frequency of p53 expression was higher than that in ALL. Bcl-2 protein immunoreactivity has been found in the majority of acute leukemia patients. The marked heterogeneity in the percentage of p53 and Bcl-2 positive cells in individual patients was observed. Comparative analysis of the distinct acute leukemia subtypes according to the percentage of p53 and Bcl-2 positive cells showed no significant differences except for p53 protein positivity in relation between T-ALL and AML cases. The samples from healthy subjects used as a control exhibited very low proportion of positively stained cells and significantly differed from p53 as well as Bcl-2 positive cases. p53 and Bcl-2 positivity have not been significantly affected neither by age, sex nor WB C counts. Association between myeloid cells maturation and proportion of p53 and Bcl-2 positive cells was observed. Noteworthy was the inverse relation between the higher proportion of p53 positive cells and low Bcl-2 positivity in some cases of acute leukemia. Although our preliminary results need to be confirmed in a larger group of patients, immunocytochemical analysis of p53 and Bcl-2 proteins, indicators of cell alterations, may help to identify risk patients requiring intensive therapy.

Reliability and limitations of cytochemistry in diagnosis of acute myeloid leukemia. Minireview.

Accurate characterization of leukemic blast cells is an important prerequisite of the precise diagnosis of acute myeloid leukemia and has a great impact on therapy and prognosis. The purpose of this review is to consider the present possibilities and limitations of enzyme cytochemistry and to emphasize how cytochemistry may contribute to the final classification and differential diagnosis of acute myeloid leukemia. The role of conventional enzyme cytochemistry, either dominant or subsidiary, in the discrimination of acute myeloid leukemia subgroups is discussed. The survey confirms the necessity of immunological marker analysis in the accurate diagnosis of minimally differentiated myeloid leukemias and acute leukemia of megakaryocytic lineage. In these cases, the cytochemical evaluation provides insufficiently relevant information regarding blast cell origin. On the other hand, cytochemical investigation is appreciated to be dominant over immunophenotyping in characterizing majority of acute myeloid leukemia subgroups, because of the availability of standardized and sufficiently specific cytochemical reactions and, because of the lack of specificity of the many of immunological markers against myeloid antigens. The immunocytochemical, cytogenetic, molecular biology and electron microscopic studies shortly mentioned in this review supplement the information for correct diagnosis of acute myeloid leukemia.

Expression of CD26 and DPP IV in T-acute lymphoblastic leukemia: comparison of immunocytochemistry with enzyme cytochemistry.

Tile possible identity ofdipeptidyl peptidase IV (DPP IV) enzymatic activity and CD26 antigen expression in phenotypically defined T-acute lymphoblastic leukemia cells (T-ALL) was examined. For comparative studies, the combination of immunocytochemistry and enzyme cytochemistry methods was used. T he strong correlation between the CD26 antigen expression and DPP IV positivity in the majority of T-lymphoblasts in T-ALL patients was evident. No CD26 antigen was expressed on DPP IV negative T-cells. The variable CD4 and/or CD8 antigen expression, frequent CD5 and CD7 positivity and absence of surface membrane CD3 antigen were the characteristic immunophenotypic features of CD26/DPP IV positive T-lymphoblasts. Moreover, the clear CD71 and CD26/DPP IV coexpression suggested the association of CD26/DPP IV positive cells with proliferation. The immunophenotype of CD26/DPP IV positive T-lymphoblasts seems to be characteristic for the relative immature cell population. In addition, noteworthy was the slight disassociation between the very high CD26 antigen expression and moderate DPP IV activity in cells of some T-ALL patients. The possible existence of enzymatically inactive structures of CD26 antigen or inactive precursors of DPP IV detectable only by immunocytochemistry was discussed. Our study indicates that CD26 antigen expression is tended to identify cells with DPP IV enzymatic activity in T-ALL patients. The results provide some more information of CD26 antigen involvement in the pathology of leukemic cells via its DPP IV enzyme activity.

CD26 and DPP IV expression in T acute lymphoblastic leukemia cells: immunocytochemistry and enzyme cytochemistry.

Recent studies have revealed the dipeptidyl peptidase IV (DPP IV) enzymatic activity of CD26 antigen. In this paper, the possible identity of DPP IV and CD26 expression in phenotypically defined T-ALL has been examined. The combination of enzyme cytochemistry and immunocytochemistry was used. The correlation between the CD26 antigen expression and DPP IV positivity in the vast majority of T lymphoblasts in T-ALL patients was observed. No CD26 was expressed on DPP IV negative T cells. The variable CD4 and/or CD8 antigen expression, frequent CD7 positivity and absence of membrane CD3 antigen expression were the characteristic immunophenotypic features of CD26/DPP IV positive T cells. CD26/DPP IV activity strongly paralleled the CD71 antigen (transferrin receptor, T cell activation/proliferation antigen) expression. The phenotypic features of CD26/DPP IV positive T cells are characteristic for the relative immature cell population. Noteworthy was the slight disassociation between the very high CD26 antigen J expression and moderate DPP IV activity in cells of some T-ALL patients. The possible existence of enzymatically inactive structures of CD26 antigen or inactive precursors of DPP IV detectable only by immunocytochemistry was discussed. Our study indicates that CD26 antigen expression is tended to identify cells with DPP IV enzymatic activity in T-ALL patients. The results provide information of CD26 antigen possible involvement in the pathology of leukemic cells via its DPP IV enzyme activity.

Immunophenotypic characteristics of T-acute lymphoblastic leukemia cells in relation to DPP IV enzyme expression.

In the present study we have examined immunophenotypic characteristics ofT-acute lymphoblastic leukemia (T-ALL) cells in relation to the expression of enzyme dipeptidyl peptidase IV (DPP IV). Peripheral blood and bone marrow cells of T-ALL patients at diagnosis were estimated. Cell surface markers were detected by a standard immunofluorescence assay and FACStar flow cytometry using a broad panel of monoclonal antibodies to define T-cell immunophenotype. DPP IV activity was investigated in phenotypically defined T-lymphoblasts. Association between DPP IV expression and proliferation was monitored by the expression of CD71 and CD38, which could be considered as markers of activation and proliferation, and by the silver-staining of nucleolar organizer regions-related proteins (argyrophilic proteins). Lymphoblasts, divided according to the presence or absence of DPP IV activity revealed remarkable heterogeneity in the immunophenotypic features. The vast majority of DPP IV positive T-ALL cases expressed CD4, CD8, CD7, CD5, CD2 along with CD71 and CD38 antigens, but the cells were surface membrane CD3 antigen negative. The phenotype of DPP IV negative cases displayed membrane CD3 antigen and variable expression of CD4 and CD8. CD71 and CD38 were frequently negative. It appears, that DPP IV active cells form the population with immature phenotype, as evidenced by mCD3 antigen absence. Relation between DPP IV positive cells and proliferation activity of T-blasts was observed, given by the presence of CD71 and CD38 positivity and overexpression of argyrophilic proteins (AgNORs). In conclusion, our study indicates a close relationship between DPP IV activity and the features ofT-cell immaturity. Association among DPP IV, CD71, CD38 and AgNORs might reflect possible relationship between immature phenotype and proliferative ability of blast cells in T-ALL patients.

The relationship between argyrophilic proteins and some immunophenotypic markers in acute leukemia cells.

This study reports the immunophenotypic features of a series of 62 selected acute leukemia patients with increased incidence of argyrophilic proteins (AgNORs) at the time of initial diagnosis. Peripheral blood and bone marrow cells of patients with T-ALL, B-precursor ALL and AML were studied. The method of silver staining was used to determine the number of AgNORs per cell. Cell surface markers were detected by a standard immunofluorescence assay. To demonstrate the relationship between AgNOR quantity and cell proliferation, the expression of activation and proliferation antigens CD38 and CD71 was investigated. To characterize the immunophenotype and the discrete stages of differentiation, the wide panel of antibodies against lymphoid, myeloid and non-lineage specific antigens was used. The number of AgNORs at diagnosis ranged from 3.05 to 6.70. Immunophenotypic analysis showed a variation in CD38 and CD71 expression among different leukemia subtypes. CD71 antigen was more expressed in T-ALL than in B-precursor ALL or in AML. Notable was the relationship between increased AgNOR quantity and antigens that characterize the immaturity of leukemic cells. The association with CD7, CD2, CD5 (without CD3 membrane expression) and CD34 in T blasts was evident. High positivity of CD19, CD10, CD34 and HLA-DR in relation to the increased amount of AgNORs in B-lineage ALL was observed. The vast majority of AML patients with high numbers of AgNORs simultaneously expressed CD13, CD33, CD34 and HLA-DR. One third of AML cases coexpressed T cell marker CD7. In conclusion, the presence of increased numbers of AgNORs at diagnosis might reflect the dependence on an early stage of leukemia cell differentiation.

The study of AgNOR proteins in leukemias: diagnostic value and correlation to S-phase cell fraction.

The study assessed the diagnostic value of silver staining method and its possible relevance as an alternative to DNA analysis for the study of cellular proliferation in various leukemias (ALL, AML, CML). Silver staining of nucleolar organizer region-related proteins (AgNORs) was applied to peripheral blood and bone marrow cells. The analysis of S-phase cells was carried out using a FACStar flow cytometer. The mean number of AgNOR dots per nucleus and the percentage of S-phase cells varied according to immunophenotype of leukemic cells, depending on the time of initial diagnosis, remission or relapse. Peripheral blood and bone marrow cells of healthy subjects exhibited less AgNOR dots than leukemic cells. The number of AgNORs in bone marrow cells was higher than that of AgNORs in peripheral blood. Significant differences between ALL and AML, as well as AML and CML in AgNOR quantity were observed. Important increase in AgNOR values was evident in relapsed leukemias and in the CML blast crisis. DNA flow cytometry analyses provided results comparable to those of AgNOR enumeration. The correlation between AgNOR dots and proportion of S-phase cells prompted us to consider that AgNOR count reflects cell proliferation capacity of leukemic cells.

Immune phenotype and some enzyme patterns in phorbol ester-induced chronic lymphocytic leukemia cells.

Leukemic cells from 10 patients with B-chronic lymphocytic leukemia (B-CLL) were isolated and cultured in the presence of 12-0-tetradecanoylphorbol 13-acetate (TPA) at a concentration of 8 x 10(-7) mol for 72 hours. Cells were analyzed before cultivation and after 72 h of cultivation with and without TPA for changes in surface membrane (Sm) and cytoplasmic (cyt) markers expression, presence of receptor for mouse rosette forming cells (MRFC) and some enzyme profiles. All B-CLL cases studied showed typical B-cell phenotype. TPA treatment induced hairy cell leukemia (HCL) characteristics, given by the membrane CD22 and CD25 expression and TRAP positivity in the majority of the cases tested. Cells had hairy cell-like morphology with more intensive cytoplasmic immunoglobulin (CIg) fluorescence staining, absent receptor for MRFC and increased activity of purine nucleosidephosphorylase. In common these changes indicate that TPA can induce hairy cell characteristics on B-CLL cells in vitro suggesting the more mature differentiation stage of HCL compared with CLL. Furthermore, we originally demonstrated that the CD22, present in the cell membrane after TPA, could be detected in the majority of unaffected B-CLL cells in their cytoplasm. From the technical point of view some intracellular CD markers and Igs of B-CLL cells in viable cells in suspension assayed by flow cytometry are described in this study.

Phorbol ester (TPA)-induced differential modulation of cell surface antigens in human pluripotential leukemia (K-562) cell line: effects of protein kinase inhibitors with broad- and PKC selective inhibitory activity.

Phorbol ester (TPA)-induced increase in cell surface expression of adhesion structures, i.e. intercellular adhesion molecule-1 (ICAM-1, CD54), beta 2 integrin LFA-1 (CD11a), complement-regulatory cell membrane protein-protein (CD59) and leukocyte common antigen (CD45) in human erythroid/myeloid leukemia cell line K-562 was inhibited by staurosporine, an inhibitor with broad, non-selective protein kinase inhibitory profile, but not by CGP 41,251, a benzoylated staurosporine derivative with the selective protein kinase C (PKC) inhibitory activity. Neither staurosporine nor CGP 41,251 modulated TPA-induced down-regulation of transferrin receptor (CD71). These data suggest that phorbol ester-induced cell surface antigen modulations in K-562 cells are predominantly mediated by PKC-independent signalling pathways.

Chronic myeloid leukemia: correlation between purine metabolism enzyme activities and membrane immunophenotype.

Peripheral blood or bone marrow of 24 patients with chronic myeloid leukemia (CML) were characterized for their surface membrane marker profiles using flow cytometry and fluorescence microscopy. Purine metabolism enzyme activities were compared with membrane immunophenotype and cytochemical stains. CML subtypes were correlated with the expression of surface membrane antigens detected by the monoclonal antibodies. On the basis of immunophenotyping we found the following characteristic marker profiles: In stable phase of CML (CML-SP)-CD15, CD11b, CDw65, CD13, in accelerated phase of CML (CML-AP)-CD15, CDw65, CD11b, CD13 and CD33, in myeloid blastic phase of CML(CML-BP-M)-CD13, CD33, HLA-DR, CD11b, CD15, CDw65, in myeloid and lymphoid (mixed) blastic phase of CML (CML-BP-M+L)-CD13, CD33, CD34, HLA-DR, CD11b, CD10 and in chronic myelomonocytic leukemia (CMML)-CD14, CDw65, CD11b, CD33 and HLA-DR. Analysis of purine metabolism enzyme activities showed that there was a correlation between the values of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) and various types of CML. ADA levels in CML-SP, CML-AP and CMML were comparable with those in normal cells. In CML-BP-M, which represents proliferation of less mature myeloid cells (similar to less mature AML subtypes), ADA activity increased and PNP activity decreased. ADA activity was significantly different between control group and CML-BP-M (p < 0.01), between CML-SP and CML-BP-M (p < 0.05). The values of PNP activity were the highest in stable phase of CML (125 pkat. 10(-6) cells) and the lowest (23 pkat.10(-6) cells) in CML-BP-M+L. PNP activity in the other groups corresponded to control values. High ADA/PNP ratio was found in CML-BP-M and CML-BP-M+L (0.7 and 2.0, respectively) in comparison to CML-SP (0.2). It follows from our results that ADA/PNP ratio enables to discriminate between stable and blast phases of CML (p < 0.01). The level of the cytochemical enzymes (CHAE, MPO, SBB, ANAE and 5' NT) varied and reflected the degree of cell differentiation and maturation. CHAE and MPO were characteristic enzymes for CML, ANBE for CMML and 5' NT for CML-BP-lymphoid.

Relation of some enzyme activities and argyrophilic proteins in childhood acute lymphoblastic leukemia.

In a series of 61 children with newly diagnosed acute lymphoblastic leukemia (ALL) the detection of argyrophilic proteins (AgNORs) in relation to enzymatic profile of leukemic blasts was undertaken. The method of silver staining was used to determine the number of AgNORs per nucleus of cells. The activity of 5'nucleotidase, acid phosphatase and beta-glucuronidase was assessed. The AgNOR proteins quantity varied with immunophenotype and cytochemical profile of leukemic cells. The enzyme 5'nucleotidase is known to be the marker enzyme in beta-precursors ALL and acid phosphatase in T-ALL blast cells. Activity of beta-glucuronidase emerged in lymphoblasts of some cases of ALL in close relation to increased number of AgNOR proteins per nucleus of leukemic cells. Our study indicates the possible importance of beta-glucuronidase involvement and increase AgNOR quantity in the proliferative activity of leukemic cells and thus they are of value in monitoring the risk groups of leukemic patients.

Ecto-5'-nucleotidase (CD73) in multidrug-resistant cell lines generated by doxorubicin.

Cytochemical screening for a panel of enzymes revealed increased 5' nucleotidase (5'NT) expression in 3 of 3 P-glycoprotein 170 (Pgp170)-positive multidrug-resistant (MDR) variants of the murine EL4 T-lymphoma cell line (EL4/ADM, ER2 and ER13). Electron microscopic localization established the presence of the membrane-bound ecto-form of the enzyme. Nine other murine, human and Chinese hamster cell lines and their MDR variants were tested for ecto-5'NT. Of these, 4 MDR variants (human cell lines MCF7A6, MCF7A2, HeLaJ2C and the murine cell line L1210A) showed increased expression of ecto-5'NT, when compared with their parental cell lines. The findings with cells of human origin were confirmed by immunofluorescent localization with a specific monoclonal antibody (MAb) (27.2) against the human ecto-5'NT. All MDR cell lines with elevated ecto-5'NT expression were generated by doxorubicin treatment. These cells were more sensitive than their parental cell lines to AMP at concentrations of 1.5-3.0 mM, confirming that the expressed ecto-5'NT was biologically active. The parental and MDR cells did not differ, in general, in their sensitivity to adenosine. An inhibitor of ecto-5'NT, alpha,beta-methyleneadenosine 5'-diphosphate, completely reversed the resistance of the EL4/ADM cell line to doxorubicin. The possibility exists of a functional relationship between the ecto-5'NT molecule and the members of the ATP-binding cassette transporter superfamily, important components of MDR, in some cell types.

Tyrosine kinase inhibitor-induced differentiation of K-562 cells: alterations of cell cycle and cell surface phenotype.

Protein tyrosine kinase (PTK) inhibitor herbimycin A inhibited proliferation, induced accumulation of cells in the G0/G1 phase of the cell cycle and a marked increae of hemoglobin-producing human leukemic K-562 cells in vitro. The isoflavonoid PTK- and topoisomerase II inhibitor genistein produced a similar effect with the accumulation of cells in the G2/M phase of cell cycle. Genistein potentiated the effect of herbimycin A on the cell cycle (i.e. decreased the proportion of S-phase cells) and induced an increased proportion of hemoglobin-producing cells. Genistein, but not herbimycin A induced a marked increase in cell surface expression of CD15 (LewisX) antigen. Both of these agents down-regulated CD45 (leukocyte common antigen) and monocyte-associated CD14 antigen on K-562 cells. Neither genistein nor herbimycin A induced increased cell surface expression of glycophorin.

Possibilities and limitations of cytochemical methods in diagnosis of acute leukemia.

Accurate identification and classification of leukemic blast cells is a very important prerequisite of the precise diagnosis of acute leukemia and has a great impact on therapy and prognosis. The purpose of this review is to consider, in the broad sense of the word, the present possibilities and limitations of enzyme cytochemistry and to emphasize how cytochemistry may contribute, on integration with the other methods of study, to the final classification and differential diagnosis of acute leukemia, a highly variable hematological disorder. In this review, the role of conventional enzyme cytochemistry, either dominant or subsidiary, in the discrimination of acute leukemia subtypes is discussed. The survey confirms the absolute necessity of immunologic marker analysis in the accurate diagnosis of acute lymphoblastic leukemia, undifferentiated or minimally differentiated leukemia and mixed-lineage leukemia because in these cases, the cytochemical evaluation provides insufficiently relevant information regarding blast cell origin, specificity of leukemia subtypes and the discrete stages of leukemic cell maturation. On the other hand, cytochemical investigation is appreciated to be dominant over immunophenotyping in characterizing acute myeloid leukemia, because of the lack of specificity of the majority of immunological markers against myeloid antigens and, because of the availability of standardized and sufficiently specific cytochemical reactions. The cytogenetic, molecular biological and electron microscopic studies mentioned in this review supplement the important information for correct differential diagnosis of acute leukemia. The prognostic impact of enzyme cytochemistry in correlation to other methods is evaluated.

Characterization of morphological and histochemical changes induced by overexpression of P-glycoprotein in mouse leukemic cell line L1210.

Vincristine sensitive (L1210) and resistant (L1210/VCR) L1210 mouse leukemia cells were studied from morphological and histochemical point of view. The morphological and histochemical findings reflected differences in membrane structure and in physiological state of sensitive and resistant cells. Numerous villous projections and cytoplasmic protrusions of the cell surface as well as higher activity of membrane enzymes (5'-nucleotidase, ATPase, alkaline phosphatase) were found in vincristine resistant cells. It is assumed that in resistant cells these differences are connected with overexpression of membrane P-glycoprotein. Moreover, in resistant cells more condensed mitochondria were found after their exposure to vincristine. This finding can reflect a higher activity of these organelles in conditions when activity of P-glycoprotein is manifested and is in agreement with increased rate of oxygen consumption by resistant cells from 2.5 +/- 0.3 to 3.3 +/- 0.2 microliter/min.10(6) cells induced by vincristine.