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BACKGROUND AND OBJECTIVE: The fibrinolytic regulator tetranectin (TN), in association with the circulating intercellular adhesive molecule-1 (cICAM-1) and interleukin -10 (IL-10), may be involved in the metastatic cascade of B-chronic lymphocytic leukemia (B-CLL). Our aim was to investigate the potential usefulness of these molecules as prognostic markers in B-CLL. DESIGN AND METHODS: Therefore, TN, cICAM-1, and IL-10 were assessed (ELISA) in the serum of 53 B-CLL patients, classified in Binet A, B, and C stages in comparison with those in 45 healthy subjects (HS). RESULTS: TN was significantly lower in B-CLL patients than in HS (9.63 [8.75-11.51] mg/L, 13.75 [12.56-14.64] ng/mL, respectively, p<10(-5)), being lower (p = 0.05) in B and C stage patients (subgroup B+C) than in A stage ones (subgroup A). cICAM-1 levels were significantly higher in B-CLL patients than in HS (475.86 [355.86-593.79] ng/mL vs. 225.62 [118.49-312.83] ng/mL, respectively, p<10(-5)) with a tendency for higher levels in subgroup B+C than in subgroup A. A significant correlation of cICAM-1 with lactate dehydrogenase (LDH) (r(s) = 0.532, p = 0.049), and a significant increase in cICAM-1 in B-CLL with diffuse bone marrow infiltration (BMI) compared to that in B-CLL with nondiffuse BMI (624.48 [557.24-726.55] ng/mL vs. 480.34 [368.96-590.34] ng/mL, respectively, p = 0.0172) were found. A significant negative correlation between TN and cICAM-1 (r = -0.5017, p = 0.0001) was observed. IL-10 was detected in all B-CLL patients and in no HS (7.37 [5.30-10.55] pg/mL), being higher (p = 0.0153) in C than in A stage patients. A significant correlation of IL-10 with TN and cICAM-1 in subgroup B+C (r(s) = -0.659 [p = 0.014] and r = 0.679 [p = 0.011], respectively) was found. CONCLUSIONS: The abovementioned findings and good performance characteristics of TN and cICAM-1 in B-CLL suggest the potential usefulness of these adhesive/recognition molecules as prognostic markers in B-CLL. The implication of these molecules along with IL-10 in the disease process deserves further study.

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A hypothesis is presented which implicates cell-cell adhesive interaction specificities in the configuration of the mode of exponential growth of acute myeloid leukemia cells in vivo. An explosion growth of the leukemic clone maintains a 1:1 ratio between proliferating and nonproliferating leukemic blasts, thus, a proliferative index (f) value of 0.5. The proposed mechanism of proliferation control is based on the expression in competent leukemic blasts (cells with capacity to proliferate) of two membrane heterodimers, of the type Ra-Li and Ri-La ("R" and "L" standing for "receptor" and "ligand" and "a" and "i", for "activation" and "inhibition", respectively). Receptor occupation and/or the resulting signal transduction is mutually exclusive between Ra and Ri on the same cell. Between cells of an interacting pair, however, intercellular contact establishing interaction of these heterodimers (through 1Ra<--2La and/or 2Ri<--1Li, numbers denoting the different cells) imposes the limitation necessary to convey, in reciprocally opposite manner, signals for activation and inhibition of the cell's proliferative activity.

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An exponential function is proposed to describe cell growth, which incorporates the parameter signifying the population's proliferative index (f). This growth function could describe with precision the increase of peripheral blasts registered in one case of untreated CML at blast crisis, in which it revealed a pattern of growth characterized by f maintained constant at 0.5. The specific rate of blast growth remained unchanged throughout CML blast crisis. Based on the proposed growth expression and the calculation of progenitor cell presence in blood, a similar pattern of growth, in which f = 0.5, became apparent for AML progenitors in two cases of relapsing AML. The presence of leukemic progenitors in blood was quantified by adopting an indirect approach. The estimated fs compared closely with 3H-thymidine indexes previously obtained (literature data) for leukemic blasts and their progenitors. It is considered that the pattern of proliferation that maintains f = 0.5 may characterize the mode of cell growth that pertains in stages of advancing leukemia. Transfer of cells from quiescence to the state of proliferative activity is assumed as controlled, viewed in the line of a model of cell growth that requires f = 0.5 and constant specific rate of growth.

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During the stage of blast crisis, the increase in the population of peripheral blasts in one examined untreated CML patient, obeyed an exponential equation of growth that requires a maintained equal proportion of proliferating to quiescent blasts. A model of cell growth at CML blast crisis is presented, which interprets the required constancy of equal-size blast subcompartments in terms of regulation of the G0----G1 flow, the latter involving activation of one cell out of three interacting quiescent blasts in contact. This model is discussed in the light of evidence that G0 blast activation involves membrane-bound interacting sites interfering with growth-promoting pathways. The model-predicted proliferative index (f) value of 0.5 +/- 0.16 is found to be nearly identical to a reported estimate of the 3H-thymidine-labeling index of CML blasts at the crisis stage of the disease. It is also close to corresponding indexes of CML blood and marrow progenitor cells and to labeling indexes of AML and ALL large blasts.

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This article reviews the authors' investigation of the enzyme RNase H (EC 3.1.4.34.) in human leukemic cells and presents the accumulated available data, based on which this enzyme is proposed to serve as a new biological parameter in the study of progression of human leukemias. The introduction gives a brief account of the occurrence, characterization and possible biological role of RNase H in cells and in retroviruses. The results reviewed briefly concern: (1) the development of a new convenient, economic and reliable assay for normal and leukemic blood mononuclear cell RNase H, which is capable of resolving subtle activity differences between samples; (2) the differentiation of RNase H levels between normal and leukemic cells; (3) the correlation of RNase H levels from different leukemia types with the severity of the disease; (4) the correlation of RNase H levels in leukemic cells with clonogenic stages in the clonal differentiation pathway; (5) the predictive potential of a RNase H activity-based parameter (phi) in assessing progression in acute myelocytic leukemia and (6) the possibility of differentiation of the RNase H levels between normal and leukemic cells via regulation of the enzyme activity at the level of antagonistic phosphorylations mediated by cAMP and calmodulin.

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1. A geometry of molecular ordering in situ characterizes the synaptosomal membrane-bound cAMP kinase, which is revealed from the expression of two equal-size [3H]cAMP-binding domains of differing sensitivity to physical (freeze-thaw) and chemical (reconstitution following salt-effected peripheral protein depletion) treatment of the membrane. 2. Cross-linking of rat synaptosomal membrane proteins with glutaraldehyde revealed after electrophoretic (agarose-polyacrylamide-SDS) resolution and "Western blot" transfer a series of bands with in situ (i.e. on the Western blots) [3H]cAMP-binding capacity. The molecular sizes of these protein bands corresponded closely with those of the cAMP kinase subunit assemblies (R2C2)2, (R2C)2, R2C2, R2C and RC. 3. The subunit assembly (R2C2)2 was also revealed after cross-linking with glutaraldehyde of the purified (commercial preparation) cytosol-derived cAMP kinase II reconstituted in lecithin liposomes. 4. The results support consideration of an operative in vivo configurational shifting between assembly forms and are discussed in the light of the outlined possibility that such shiftings might be involved in the regulation of the membrane-bound cAMP kinase.

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A new biological parameter (ψ) has been obtained and proposed here to serve in the assessment of acute myeloid leukemia (AML) progression. It is a function of the activity of RNase H (EC 3.1.4.34), the latter determined in mononuclear cells from the peripheral blood of AML patients. Using a series of patients at the time of diagnosis and after 1-2 cycles of chemotherapy, the enzyme was assayed before the several times during chemotherapy. The derivation of 4 was based on evidence suggesting that the enzyme level correlates with the proliferating leukaemic blasts and their progenitors. Values ψ>1 signify the presence of clonogenic leukaemic progenitor cells in the peripheral circulation. When these high (> 1) ψ values were found during chemotherapy, in these cases it was possible to predict an increase of the peripheral blast pool, with 82% success, occurring 5-35 days before cytologic relapse. In the patients in whom at some stage during treatment, ψ acquired values above unity or in whom ψ increased progressively, survival time was in linear correlation with the time period from the initiation of treatment to the documentation of this high ψ estimate. These results suggest that a patient's relapse risk can be defined by ψ with some degree of precision.

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Peripheral blood mononuclear cells (PBMNC) from 23 healthy subjects and 39 patients with B-cell chronic leukaemia (B-CLL) were assayed for ribonuclease H activity using as substrate the filter-immobilized synthetic homopolymer hybrid 3H-poly(rA):poly(dT). In 69% of the leukaemia patients examined enzyme activities were above those estimated in cells from the healthy controls. The mean enzyme levels for the normal and the leukaemic samples group were (per cent substrate hydrolysis): 8.1 +/- 8.9 and 58.7 +/- 40.8, respectively, their difference being statistically highly significant (P less than 0.0001). This result does not represent homogeneity within the cells but is due to a subclass of cells within the leukaemic clone containing the enzyme, thus contributing through pool size fluctuation to the wide variations of enzyme activity observed among the patients. These cells containing high activity could not be identified with either the prolymphocytes or the large lymphocytes. The activity of ribonuclease H in the examined CLL patients correlated with disease stage (Binet) (P = 0.011) and appeared to serve as a sensitive indicator of disease progression when compared with a number of other known prognostic parameters.

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1. The binding of [3H]cAMP in vitro to synaptosomal membranes from rat brain was resolved in two components; one saturable at 20 nM cAMP with dissociation constant (KD) of 4.7 nM, and another nonsaturable within the 5-133 nM cAMP concentration range with an estimated KD value of 0.26 microM. 2. MgATP at concentration of 0.4 mM effected complete inhibition of the binding of [3H]cAMP to synaptosomal membranes throughout the used concentration range. This and the above finding indicate that the studied binding was focused on to the cAMP kinase on the membrane. 3. Calcium at concentrations of 0.1 and 10 mM stimulated a transient 20-30% increase of [3H]cAMP binding to the membranes which was influenced, as regards its time of appearance, by the concentration of cAMP. 4. The stimulation by calcium of the binding of [3H]cAMP to the membranes was inversely related to the phosphorylation of an Mr = 80,000 membrane protein, indicating stimulation of a negative effector function of cAMP--through cAMP-mediated phosphorylation--in the phosphorylation by calcium of this substrate. Moreover, the temporal displacement by cAMP of the peak of [3H]cAMP binding, produced similar temporal displacement of the inhibitory effect of cAMP on the Mr = 80,000 substrate phosphorylation. 5. These results suggest interaction in vitro of calcium and cAMP in modulation of the activity of cAMP kinase on the synaptosomal membranes.

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The activity of hybrid ribonuclease (ribonuclease H) has been determined in mononuclear blood cells (lymphocytes plus monocytes) from 23 normal individuals and cells (pool of immature granulocytes, metamyelocytes and lymphocytes) from 35 untreated acute and chronic myelogenous leukemia cases. It was found that in 86% of the leukemic samples the activity of ribonuclease H was above two standard deviations from the mean activity level drawn for the group of normal samples along the 0-100% substrate hydrolysis scale. The activity of the enzyme in leukemic cells correlated linearly with the DNA-synthesizing activity of the cells in vitro and in the examined CML cases it paralleled the inverse relationship of the incorporation of tritiated thymidine into DNA to the size of the pool of immature granulocytes. In one CML patient who received chemotherapy with Myleran, the activity of ribonuclease H, high at the initiation of drug therapy, was reduced to a normal level at remission, but increased again at the stage of subsequent relapse. These findings indicate that the levels of ribonuclease H in leukemic cells reflect the proliferative activity of the population in the cases of untreated myelogenous leukemias.

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The tissue responds in culture differently to each of the tested hormones. Insulin, but not prolactin or hydrocortisone, was mitogenic to the culture, but did not alter the overall transcriptional activity pattern displayed by the insulin-free cultures. Prolactin stimulated transcriptional activity above control levels between days 2 and 6 of culture, but hydrocortisone was inhibitory to both DNA-synthetic and transcriptional activity of the culture. Upon initial 24-hr treatment with 50 micrograms/ml monovalent concanavalin A, the tissue altered its hormonal sensitivity in the examined activities, but the presence of any one of the hormones in the culture medium abolished the individual effect of concanavalin A. With hydrocortisone, however, the individual effects of the lectin and the hormone interacted in cultures treated with both compounds, suggesting a type of intracellular communication with the membrane, operative in this mammary cell line, that may involve channels of biochemical changes energized by a hydrocortisone stimulus.

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A method for assaying hybrid ribonuclease has been devised which utilizes as substrate the synthetic hybrid [3H]polyriboadenylic acid [poly(rA)]:polydeoxythymidylic acid [poly(dT)] immobilized on the solid matrix of nitrocellulose filters. The hybridization on filter of [3H]poly(rA) to poly(dT) has been explored in terms of efficacy of the process and the response of the product to RNase H. A pulse of uv irradiation of poly(dT) while in dry state on the filter increased its firm binding to the filter in a concentration-dependent manner, resulting in a concomitant increase of the yield of hybrid formation. The filter-immobilized hybrid was 95% resistant to RNase A but sensitive to RNase H. When stored in toluene in the cold the hybrid maintained its stability for over 6 months, as judged by its resistance to RNase A. The method offers a number of advantages over assays that use solution hybrids as substrates and was readily applicable in the screening of leukemic patients, in the leukocytes of which it has demonstrated increased RNase H levels.

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Insulin induced in organ culture of a mouse mammary adenocarcinoma the expression of two cytoplasmic proteins of molecular size 72 and 49 kD. In freshly excised tumor both proteins are present in relatively large amounts, but their concentration became greatly diminished during culture in insulin-free media, indicating sensitivity to post-transcriptional modification which was activated in vitro in the absence of insulin. In insulin cultures the two proteins reappeared after two culture days, their concentration becoming increased thereafter with time of culture, but their expression could not be correlated with the biphasic pattern of transcriptional activity fluctuation recorded during culture similarly to insulin-free cultures. Insulin on the other hand stimulated two waves of doubling of DNA synthesis during 6 culture days. The results demonstrate insulin responsiveness in culture of a proliferative fraction of the tumorous cell population in this adenocarcinoma line in the synthesis of two sensitive proliferation-derived cytoplasmic proteins. The possible biological significance of these proteins is discussed.

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Protein phosphorylation in cerebral cell-free preparations from neonate rabbits was inhibited by bilirubin and promoted by aminophylline when these substances had been administered intravenously. In animals given both compounds, the bilirubin-induced inhibition of phosphorylation was partly reversed by aminophylline. Adenosine 3',5'-monophosphate added in vitro during the assays also increased protein phosphorylation. These data introduce new concepts in the pathogenesis of kernicterus.

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The 'binding' of cholesterol on to dog brain synaptosomal plasma membranes from aqueous cholesterol 'solutions' was studied. 'Binding' of exogenous cholesterol is a slow process, strictly depending on the concentration of cholesterol and the quantity of the membranes present. It appears that binding probably occurs in three distinct successive stages. The first stage occurs very rapidly, and consists of a large deposition-like accumulation of cholesterol onto the membranes. This stage is characterized by the lack of functional changes of integral proteins. It is followed or accompanied by a slower type of binding, probably at 'specific binding sites', the nature of which is, in all probability, cooperative. Thus, when the glucoside of cholesterol is used at lower concentrations as compared to cholesterol it increases the binding of cholesterol, while at higher concentrations relative to cholesterol, it antagonizes its binding. This stage, which evokes strong functional changes of integral proteins, merges without interruption into an incorporation of cholesterol as a structural element into the membranous framework (nonspecific binding).

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This study examines certain aspects of the interaction of acridines with DNA. A comparative study of the methods available for the determination of the association constants (Kap) for compounds which interact with DNA has been pursued. A new equation which permits the spectrophotomeric determination of Kap has been derived. This equation can be applied to compounds which upon interaction with a polymer exhibit discrete absorption changes. Application of this equation to substituted acridines and tetrahydroacridines yields some preliminary information on the effect of ring substituents on the interaction of acridines with DNA. Low levels of DNA/dye ratios have been used in the studies reported herein.

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