Thermochemical scanning probe lithography of protein gradients at the nanoscale.

Patterning nanoscale protein gradients is crucial for studying a variety of cellular processes in vitro. Despite the recent development in nano-fabrication technology, combining nanometric resolution and fine control of protein concentrations is still an open challenge. Here, we demonstrate the use of thermochemical scanning probe lithography (tc-SPL) for defining micro- and nano-sized patterns with precisely controlled protein concentration. First, tc-SPL is performed by scanning a heatable atomic force microscopy tip on a polymeric substrate, for locally exposing reactive amino groups on the surface, then the substrate is functionalized with streptavidin and laminin proteins. We show, by fluorescence microscopy on the patterned gradients, that it is possible to precisely tune the concentration of the immobilized proteins by varying the patterning parameters during tc-SPL. This paves the way to the use of tc-SPL for defining protein gradients at the nanoscale, to be used as chemical cues e.g. for studying and regulating cellular processes in vitro.

Influence of sorbitol on protein crowding in solution and freeze-concentrated phases.

Small-angle neutron scattering was employed to study protein crowding under freezing conditions that mimic those used in pharmaceutical processing. The results demonstrate that, although there is an increase in heterogeneity as the temperature is reduced, sorbitol reduces protein crowding in both solution and freeze-concentrated phases, thus protecting the protein from forming oligomers or irreversible aggregates.

Atomistic ensemble modeling and small-angle neutron scattering of intrinsically disordered protein complexes: applied to minichromosome maintenance protein.

The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea and eukarya. In this work we determined the solution structure of the N-terminal portion of the MCM complex from the archaeon Methanothermobacter thermautotrophicus (N-mtMCM) in the presence and absence of DNA using small-angle neutron scattering (SANS). N-mtMCM is a multimeric protein complex that consists of 12 monomers, each of which contains three distinct domains and two unstructured regions. Using an all-atom approach incorporating modern force field and Monte Carlo methods to allow the unstructured regions of each monomer to be varied independently, we generated an ensemble of biologically relevant structures for the complex. An examination of the subsets of structures that were most consistent with the SANS data revealed that large movements between the three domains of N-mtMCM can occur in solution. Furthermore, changes in the SANS curves upon DNA binding could be correlated to the motion of a particular N-mtMCM domain. These results provide structural support to the previously reported biochemical observations that large domain motions are required for the activation of the MCM helicase in archaea and eukarya. The methods developed here for N-mtMCM solution structure modeling should be suitable for other large protein complexes with unstructured flexible regions.

Antibodies in human sera to oncorna virus-like proteins from normal or leukemia marrow cell cultures.

Some human marrows in culture release particles with oncornavirus-like properties. This study was designed to examine the immunological properties of similar particles in human marrow culture supernates. Leukemic and nonleukemic marrows were cultured for 5-7 days in the presence of [14C]uridine and [3H]leucine or [3H]glucosamine. Labeled supernatant components banding in sucrose gradient densities of 1.20-1.24 g/ml were used as antigen in a double antibody immunoprecipitation assay. The assay was validated by end point titrations and competition with unlabeled antigen; purified myeloma proteins were used as negative controls. Cross-reactivity with mammalian oncornaviruses, as judged by competitive inhibition of precipitation by these viruses, was slight and at the border of the sensitivity of the method. Precipitated antigens analyzed by SDS polyacrylamide gel electrophoresis contained three distinct polypeptides of about 70,000, 45,000 and 30,000 mol wt; these comigrated with the gp 70, pg 45, and p 30 of a murine leukemia virus. Similar polypeptides were obtained from both leukemic and nonleukemic marrow culture supernates. As determined by the radioimmunoprecipitation assay, 32 of 45 leukemic sera (71%), 36 of 45 normal sera (80%), 15 of 19 sera from family contacts of leukemic patients (79%), 14 of 21 cord blood specimens (67%), and 21 of 23 sera (91%) from patients with systemic lupus erythematosus had detectable antibody activity.

Inhibition of human factor VIIIa by anti-A2 subunit antibodies.

Human inhibitory alloantibodies and autoantibodies to Factor VIII (FVIII) are usually directed toward the A2 and/or C2 domains of the FVIII molecule. Anti-C2 antibodies block the binding of FVIII to phospholipid, but the mechanism of action of anti-A2 antibodies is not known. We investigated the properties of a patient autoantibody, RC, and a monoclonal antibody, 413, that bind to the region which contains the epitopes of all anti-A2 alloantibodies or autoantibodies studied to date. mAb 413 and RC were noncompetitive inhibitors of a model intrinsic Factor X activation complex (intrinsic FXase) consisting of Factor IXa, activated FVIII (FVIIIa), and synthetic phospholipid vesicles, since they decreased the Vmax of intrinsic FXase by > 95% at saturating concentrations without altering the Km. This indicates that RC and mAb 413 either block the binding of FVIIIa to FIXa or phospholipid or interfere with the catalytic function of fully assembled intrinsic FXase, but they do not inhibit the binding of the substrate Factor X. mAb 413 did not inhibit the increase in fluorescence anisotropy that results from the binding of Factor VIIIa to fluorescein-5-maleimidyl-D-phenylalanyl-prolyl-arginyl-FIXa (Fl-M-FPR-FIXa) on phospholipid vesicles in the absence of Factor X, indicating it does not inhibit assembly of intrinsic FXase. Addition of Factor X to Fl-M-FPR-FIXa, FVIIIa, and phospholipid vesicles produced a further increase in fluorescence anisotropy and a decrease in fluorescence intensity. This effect was blocked completely by mAb 413. We conclude that anti-A2 antibodies inhibit FVIIIa function by blocking the conversion of intrinsic FXase/FX complex to the transition state, rather than by interfering with formation of the ground state Michaelis complex.

Hemopoietic colony growth-promoting activities in the plasma of bone marrow transplant recipients.

Plasma samples were obtained from 34 bone marrow transplant (BMT) recipients before and after administration of the preparative regimen and tested for their ability to promote and/or support growth of hemopoietic colonies. The ability of plasma samples to promote colony formation on their own was tested on normal nonadherent target cells without addition of exogenous growth factors. The growth-supporting activity was examined in the presence of medium conditioned by phytohemagglutinin-stimulated leukocytes (PHA-LCM) and/or erythropoietin (EPO). A series of kinetic changes was routinely observed. Pretransplant samples rarely gave rise to colonies without addition of exogenous growth factors. Plasma samples obtained after completion of the preparative regimen demonstrated increments of growth-promoting activities for megakaryocyte and granulocyte-macrophage progenitors (CFU-Meg and CFU-GM), respectively, that peaked between 7 and 21 d after transplantation. By day 30, activity levels of some patients had returned to pretransplant values, whereas in other patients, activities remained elevated. Persisting activity levels were associated with delayed engraftment. In contrast, activities for progenitors committed to erythropoiesis (BFU-E) and pluripotent precursors (CFU-GEMM) were only rarely observed. The activities were independent of febrile episodes. Their growth-promoting influence on CFU-GM could be neutralized completely by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies. These data suggest that at least some of the observed activities in post-BMT plasma are related to GM-CSF. The growth-supporting activities of pretransplant plasma samples are lower than normal plasma when tested on CFU-Meg and CFU-GM. The growth-supporting activities improved transiently within the first month after BMT. A decline during the second and third month was followed by a gradual return to activity levels that were comparable to normal plasma. The effects of these plasma samples on BFU-E and CFU-GEMM were assessed with PHA-LCM and EPO. Similar to CFU-Meg- and CFU-GM-supporting capabilities, they improved transiently after BMT with a return of normal support function after 5-6 mo. The observed endogenous production of growth-promoting and growth-supporting activities for hemopoietic progenitors may serve as a background to design clinical trials for the timely administration of recombinant hemopoietic growth factors to BMT recipients.

Self-renewal capacity of leukemic blast progenitor cells.

A review is presented of experimental information pertaining to the characteristics of a procedure designed to quantitate the capacity for self-renewal in clonogenic cells of human acute myeloblastic leukemia. In a series of 44 previously untreated patients, a significant correlation (p less than 0.01) was seen between low capacity for self-renewal and successful remission induction. Three cytotoxic drugs (Adriamycin, 1-beta-D-arabinofuranosylcytosine, and N-[4-(19-acridinylamino)-3-methoxyphenyl]-methanesulfonamide) were tested for preferential effect against self-renewal events. Surviving clonogenic cells to these agents had, respectively, unchanged, lower, and higher capacity for self-renewal. The implications of such drug properties are discussed.

High-dose cytosine arabinoside in the treatment of acute myelogenous leukemia: contributions to outcome of clinical and laboratory attributes.

High-dose cytosine arabinoside (HDAra-C) has been used for remission induction, and in conventional doses for maintenance in a trial of single-agent therapy in 43 previously untreated patients with acute myelogenous leukemia (AML). Rationale for the trial was provided by the observed decrease in leukemic blast cell self-renewal in culture following exposure to Ara-C. Compared with a previous trial of 57 patients treated with multidrug therapy, single-drug Ara-C was associated with a significantly improved complete remission rate (P = .010), although the survival time was not increased. All patients with low self-renewal responded to HDAra-C in contrast to the previous trial where some patients with this phenotype failed remission induction. The clinical observations are consistent with the view that the antileukemic effect of Ara-C has some specificity for cellular events required for self-renewal of blast cells. Exposure in vivo to Ara-C was associated with an increase in blast stem cell renewal at relapse, indicating that maintenance with other drugs should be tested. The study demonstrates the importance of biological attributes in design and analysis of clinical trials.

Contributions of host- and disease-related attributes to the outcome of patients with acute myelogenous leukemia.

Three sequential trials of treatment for acute myelogenous leukemia (AML) involving 173 patients were analyzed to identify clinical and myeloblast-cell progenitor properties in culture related to outcome. The latter, including self-renewal capacity expressed as plating efficiency (PE2) and drug sensitivity, were determined for a representative group of 45 patients. Despite increasingly intensive remission induction therapy, similar response rates were achieved in the three trials and no increase in the duration of survival was observed. Clinical attributes at presentation by multivariate analyses were not consistently predictable of outcome. Of the blast cell attributes, only PE2 was predictive of duration of survival (p less than 10(-6)). For patients in remission the relapse rate during the first year was 0.63 compared with 0.15 in subsequent years. The percentage marrow myeloblasts at presentation, a measure of disease activity, was significantly higher for the patients having remissions lasting less than one year. These studies demonstrate the importance of disease-related attributes on the outcome of patients with AML.

Prognostic groups for management of localized Hodgkin's disease.

Two hundred fifty-two patients receiving radical irradiation for clinical stages I and II Hodgkin's disease between 1968 to 1977 had an actuarial ten-year survival rate of 78% and a relapse-free rate of 61%. Sixty-seven patients receiving chemotherapy followed by radiation had a 78% survival rate and a 63% relapse-free rate. Independent prognostic factors for survival and relapse were age, stage, and histology. Disease bulk was predictive only of relapse. Neither site of presentation above or below the diaphragm nor presence of mediastinal involvement was predictive for survival or relapse; however, patients with large mediastinal masses (greater than or equal to 10 cm absolute diameter) had a significantly higher intrathoracic failure rate with conventional mantle irradiation. Analysis of failure, according to age, clinical stage, and histologic type, showed three groups of patients defined according to the risk of relapse with radiation therapy: those with isolated upper cervical stage IA disease (group 1, relapse rate 8%), younger patients with localized stages I and II disease of favorable histologic type (group 2, relapse rate 35%), and older patients with extensive or symptomatic stages I and II disease of less favorable histologic type (group 3, relapse rate 70%). Subsequent analysis of radiation treatment volume indicates that the use of upper abdominal irradiation for patients in group No. 2 could yield results equivalent to those achieved with radiation therapy for surgically staged patients.

Sensitivities of AML blast stem cells to idarubicin and daunorubicin: a comparison with normal hematopoietic progenitors.

The objective of this study was to determine whether or not cell culture studies could contribute to the correct choice between idarubicin (IDA) and daunorubicin (DNR) for combination with ara-C in remission induction therapy of AML. Two growth factor-sensitive AML cell lines and the peripheral blood blast cells from 10 patients with AML were studied in culture for sensitivity to IDA and DNR under four culture conditions; cells were grown either in methylcellulose or suspension culture in the presence of G-CSF or GM-CSF. Normal bone marrow cells were cultured in methylcellulose in the presence of IDA or DNR under conditions suitable for the detection of BFU-E and CFU-C. Dose-response curves for AML blast cells were characterized by an initial shoulder and then an exponential decrease in survival. Marked patient-to-patient variation was observed for both portions of the survival curves. IDA was significantly more toxic to blast cells than DNR, especially for more sensitive cell populations. Consistent differences in drug sensitivity in the four culture conditions were not observed. BFU-E and CFU-C dose-response curves of normal marrow progenitors resembled those of AML blast cells but in contrast fell within a narrow range. The culture studies support the clinical finding in AML of modest superiority of IDA over DNR. The heterogeneity in sensitivity of AML blasts in culture suggests an opportunity to individualize treatment. Preclinical studies may help in developing such a therapeutic approach.

Interaction between retinoic acid and cytosine arabinoside affecting the blast cells of acute myeloblastic leukemia.

Retinoic acid (RA) is a potent morphogen that has been shown to increase differentiation in some leukemic cell populations. RA has been used in treatment of some patients with acute myeloblastic leukemia (AML) and myelodysplastic syndromes. In previous experiments we had observed that RA may decrease the self-renewal of blast cells in established cell lines, and in our clinic RA has been tested as maintenance treatment in association with chemotherapeutic drugs. Accordingly, we asked if exposure of AML blast cells to RA affected their subsequent response to ara-C. We found that brief exposure to RA regularly increased the ara-C sensitivity of cells from two established AML cell lines. A similar, though less marked, effect was seen when the blast cells from one patient were tested directly; in a second instance, highly ara-C resistant blasts did not become sensitive when exposed to RA. Experiments using high specific activity tritiated thymidine did not disclose any changes in the proportion of AML cells in the DNA synthesis phase of the cycle at times when their responses to ara-C were changing. We interpret our findings as support for continuing efforts to integrate RA in the management of AML patients and suggest that the mechanism of ara-C sensitization may not depend on changes in the cell cycle.

Granulocyte-macrophage colony-stimulating factor and interleukin-3 protect leukemic blast cells from ara-C toxicity.

The blast cells of acute myeloblastic leukemia (AML) usually require growth factors for optimum proliferation in cell culture. Growth factors also affect the sensitivity of AML blast cells to cytosine arabinoside (ara-C). Others have reported that factor-treated cells are more ara-C sensitive than blasts in culture without factors. These authors have reported previously that AML blasts grown with rG-CSF, with or without GM-CSF, are more sensitive than cells in GM-CSF alone. This paper reports experiments which show that changes in the ara-C sensitivities of blast cells in different growth factors are not explained by changes in the percentage of cells in the DNA synthesis (S) phase of the cycle. Blasts freshly obtained from five AML patients were cultured in either rG-CSF, rGM-CSF, or rIL-3; they were then exposed to 20 min pulses of either high specific activity tritiated thymidine (3HTdR) or a high concentration of ara-C. Regardless of the factor present, the pulse of 3HTdR decreased the number of clonogenic cells by about 50%, the result expected for actively proliferating cells with an S phase occupying about half the cycle time. The same result was found for four of the five blast cell populations grown in G-CSF and pulsed with ara-C; in contrast, clonogenic cells grown in GM-CSF or IL-3 from these four populations were not killed by ara-C. The blasts from the fifth patient were ara-C resistant under all conditions. It was concluded that exposure to GM-CSF or IL-3 decreased ara-C sensitivity in blasts that were actively making DNA. The observation was explored in more detail using a cell line (OCI/AML-1a) that is both ara-C sensitive and growth factor dependent. These studies showed that about 15 h of growth in factor are required for a change in ara-C sensitivity.

Comparison of the effects of all-trans and cis-retinoic acid on the blast stem cells of acute myeloblastic leukemia in culture.

Recent work has shown that acute promyelocytic leukemia (APL) cells have a characteristic translocation involving the retinoic acid receptor on chromosome 17 and the myl protein on chromosome 15. Patients with APL respond to the administration of all-trans-retinoic acid. A cell line with t15;17 (NB4) has recently been reported; this line responds to all-trans-retinoic acid with differentiation. There is also a recent report showing that all-trans-retinoic acid is more active than cis-retinoic acid in inducing differentiation in freshly obtained APL cells. All-trans-retinoic and cis-retinoic acid are compared for their effects on growth in culture of freshly obtained AML cells, cell lines without t15;17, and NB4 cells. While all of these AML populations responded to both forms of retinoic acid, NB4 cells only were much more sensitive to all-trans-retinoic acid compared to cis-retinoic acid. The difference was seen when the NB4 cells were exposed in suspension and not when colony-formation in methylcellulose was used as an end point. Both forms of retinoic acid increased the sensitivity of blast cells to cytosine arabinoside; for NB4 cells, the sensitization was much greater when all-trans-retinoic acid was used rather than cis-retinoic acid. We conclude that the increased effects of all-trans-retinoic acid are specific for APL cells, and that a major effect of retinoic acid is on blast stem cell self-renewal.

Expression of a retinoic acid receptor gene in myeloid leukemia cells.

Retinoic acid (RA) has been shown to increase differentiation in some leukemia cell lines (HL-60 and KG-1) but not others (K562). Similarly, RA has been reported to have variable effects on fresh blast cells. Recently, molecular clones have been obtained for the nuclear receptor for retinoic acid. The experiments described in this paper were designed to compare expression of the receptor to biological activity in myeloblastic leukemia cells. In four continuous AML cell lines, a positive correlation was found between retinoic acid receptor (RAR) expression by Northern analysis or RNA dot blot and the ability of RA to inhibit colony formation. Kinetic studies of the most sensitive cell line showed that inhibition of colony formation was associated with reduced blast cell self-renewal and differentiation-like events. RAR was detected in freshly obtained blast cells from 23 AML patients. Patient-to-patient variation was observed; however, a correlation was not found between RAR expression and response of the freshly obtained blast cells to RA.

Response to 5-azacytidine of leukemic blast cells in suspension: a biological parameter associated with response to chemotherapy.

Sensitivities to drugs acting on cells in culture can be measured as dose-response curves, provided a quantitative assay is available for a relevant cell function. We have used two such assays in the study of the blast cells of acute myeloblastic leukemia. Colony formation in culture with methylcellulose detects principally terminal divisions, while growth of clonogenic cells in suspension reflects self-renewal. In a previous study different cytosine arabinoside and 5-azacytidine dose-response curves were obtained with the two assays. For the former the slope of the dose-response curve measured in suspension was steeper than that obtained using the clonogenic assay. For the latter, 5-azacytidine, the relationship between sensitivity in suspension and in methylcellulose was reversed. Further, for cytosine arabinoside, sensitivity in suspension but not in methylcellulose was associated with successful remission-induction. In this article we report an association between 5-azacytidine sensitivity in suspension and successful remission induction, for patients treated only with high-dose cytosine arabinoside. There was no correlation between the 5-azacytidine dose-response curve in methyl-cellulose and clinical outcome. A model is presented that may explain these findings, based on the hypothesis that there are genetic mechanisms responsible for blast cell renewal.

Cytosine arabinoside (ara-C) and cis-dichlorodiammineplatinum II (cisplatin) alone and in combination: effects on acute myeloblastic leukemia blast cells in culture and in vivo.

Cytosine arabinoside (ara-C) and cis-dichlorodiammineplatinum II (cisplatin) are lethal to mammalian cells by very different mechanisms; however, they share interactions with the biology of blast cells in acute myelogeneous leukemia (AML). Both agents are more toxic to AML blasts in suspension than when a clonogenic assay in methyl cellulose is used; both agents are more toxic in suspension in the presence of rG-CSF than with rGM-CSF. Accordingly, preclinical tests were undertaken of cisplatin and ara-C in combination. At the same time, a phase I/II clinical trial of the combination was conducted, using AML patients refractory to treatment or in relapse. In the laboratory, blasts from eight AML patients were tested against each agent singly and in combination. The observed survival values for the mixture were compared with those predicted by assuming either an additive effect or a more general effect that allows synergism or antagonism. Blasts from two patients were tested with this design in the presence of rG-CSF or rGM-CSF. In most instances the toxic effects of ara-C and cisplatin were additive. Evidence of synergism was seen in blasts from three patients.

The expression of the proto-oncogene C-kit in the blast cells of acute myeloblastic leukemia.

The oncogene kit has been shown genetically to map in the W locus of the mouse. This locus is known to have an important role in the regulation of normal hemopoietic stem cell growth. The blast cells of acute myeloblastic leukemia may be considered to arise in predeterministic stem cells. Accordingly, we sought evidence that kit was involved in the regulation of AML blast growth, using a cDNA probe to the external domain of c-kit. With this probe the gene was found to be in germline configuration in blast cells from AML, ALL, and continuous myeloblastic cell lines. However, expression could be detected by Northern analysis or RNA dot blots only in fresh AML blast cells. Fresh cells from ALL patients, normal bone marrow, PHA-stimulated lymphocytes, and four myeloblastic continuous cell lines were expression negative by the same techniques.

High-dose cytosine arabinoside remission induction for acute myelogenous leukemia: comparison of two regimens of remission maintenance.

We report a single institution sequential trial of two maintenance treatment regimens for patients with acute myelogenous leukemia (AML). A total of 175 consecutive patients with AML received initial remission induction therapy with high-dose cytosine arabinoside (ara-C) and glucocorticoids. For the initial 63 patients (group A), the control population, planned maintenance treatment was with conventional-dose ara-C given over 4 days for up to 18 months. The subsequent 107 patients (group B) had planned maintenance therapy of up to 6 courses of daunorubicin, ara-C and prednisone and daily cis-retinoic acid for up to two years. The presenting features of group A and B patients were similar as were the response to remission induction, 60 and 52%, respectively. Severe neurological toxicity was encountered once after high-dose ara-C; no drug-related deaths occurred during maintenance treatment. Median duration of remission for group B patients was 9.9 months compared with 5.5 for group A (p = 0.0685). Median survival duration for the two groups was similar, 9.1 months for group A and 10.4 for group B. Survival of patients in group B who attained a complete remission was significantly better than that of patients in group A (p = 0.0439). The studies confirm our initial experience with remission induction using single agent high-dose ara-C and suggest a positive role for maintenance therapy in AML.

Heterogeneity in acute myeloblastic leukemia.

Morphological-identified blast populations are the hallmark of the malignant clones that dominate hemopoiesis in acute myeloblastic leukemia (AML). Marked heterogenity is characteristic of AML blasts. Patient-to-patient variation is seen in their biological properties but is particularly evident in the response to treatment. Intraclonal variation is generated during clonal expansion, particularly as blast stem cells either undergo self-renewal or enter into a series of terminal divisions. These two alternative activities can be monitored in cell culture using a clonogenic assay and a suspension assay. The balance between renewal and differentiation can be altered by exposing blast populations to various growth factors in culture. Further, certain drugs, particularly ara-C, appear to be more toxic for self-renewing divisions than cell-cycle events generally. We suggest that both drugs and growth factors should be assessed for their effects on self-renewal as part of preclinical testing.