Expression on human thymocytes of the idiotypic structures (Ti) from two leukemia T cell lines Jurkat and HPB-ALL.

The expression on a significant number of thymocytes of idiotypic structures (Ti) restricted to HPB-ALL or Jurkat cells is demonstrated. As many as 2-4% of thymocytes were stained with anti-Ti HPB-ALL or anti-Ti Jurkat monoclonal antibodies, when analyzed by flow microfluorometry. Immunohistochemical localization studies performed on frozen thymus specimens of either fetal or pediatric origin indicated a scattered distribution of Ti-positive cells in both the cortex and the medulla. From lysates of 125I-labeled pediatric thymocytes, anti-Ti HPB-ALL and anti-Ti Jurkat monoclonal antibodies precipitated disulfide-linked heterodimers comparable to those precipitated from 125I-labeled HPB-ALL or Jurkat cells as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

Purification and characterization of actin from normal and chronic lymphocytic leukemia lymphocytes.

Previous studies from this laboratory have shown actin to be a major protein of human lymphocytes (Stark, R., Liebes, L. F., Nevrla, D., and Silber, R. Biochem. Med., 27: 200-206, 1982). We now report the purification to homogeneity and characterization of actin from blood lymphocytes of normal subjects and patients with chronic lymphocytic leukemia. The recovery of the purified protein was about 20%. The properties of the lymphocyte actins were compared to each other and to those of rabbit skeletal muscle actin. Lymphocyte actin consisted of beta and gamma forms in a 2:1 ratio. The Mr 42,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Normal and leukemic lymphocyte actin had similar polymerization properties as assessed by viscosity measurements at 25 degrees and 4 degrees, and the ultrastructural appearance of the filaments was the same. Similar patterns were observed between normal and chronic lymphocytic leukemia actin tryptic digests analyzed by high-performance liquid chromatography. The Vmax of the actin-activated myosin Mg2+ ATPase activity was compared using rabbit skeletal muscle heavy meromyosin and subfragment 1 preparations. The values obtained with rabbit skeletal muscle and normal lymphocyte actin were identical. The Vmax observed with chronic lymphocytic leukemia lymphocyte actin was 70% of that obtained with normal lymphocyte actin. The amount of actin needed to produce half-maximal activation (Kapparent) of heavy meromyosin and subfragment 1 were, respectively, 26 and 25 microM for normal lymphocytes and 18 and 24 microM for chronic lymphocytic leukemia lymphocytes. The anomalous ATP activation by actin did not reflect differences in B-:T-cell subpopulations between chronic lymphocytic leukemia and normal lymphocytes. The possible significance of the observed differences between the myosin Mg2+ ATPase activation by chronic lymphocytic leukemia and normal lymphocyte actin is discussed.

Analysis of human leukemic cells by use of high-resolution two-dimensional electrophoresis. I: results of a pilot study.

We analyzed mononuclear leukocytes from patients with various human leukemias by high-resolution two-dimensional electrophoresis. Tumor cells of the granulocytic, monocytic, and lymphoid lineages [obtained from chronic granulocytic leukemia in blast transformation, acute monocytic leukemia, and chronic lymphocytic leukemia (CLL), respectively] can be easily recognized by using a series of cell-type marker proteins identified by comparison of fractionated normal cell populations. B and T cell types of CLL could be distinguished, the results correlating well with those obtained by use of monoclonal-antibody staining methods. In two cases representing almost pure B-cells (classical CLL; 0% T, 85% B) and T-cells (cutaneous T-cell leukemia; 77% T, 0% B), 27 of 29 marker proteins showed quantitative B/T differences comparable to those observed in comparisons of normal B-and T-lymphocytes prepared by cell sorting. These results indicate that cells from relatively well-differentiated leukemias show complex patterns of gene expression very similar to those of the corresponding normal cells and strongly support the use of large marker panels in cell-type determination. Less-well-differentiated acute leukemias [such as acute undifferentiated and acute granulocytic (FAB:M1)] appear to yield protein patterns corresponding less closely to recognizable mature cell types, and may show expression of novel proteins related to the state of differentiation.

[Urinary and fecal excretion of porphyrins and their precursors in leukemia patients]. / Prouchvane na izluchenite s urinata i izprazhneniiata porfirini i niakoi tekhni predshestveitsi pri levkozno bolni.

Delta-aminolevulinic acid (DALA), porphobilinogen (PBG) and porphyrins in urine and feces were determined in 40 healthy controls (20 males and 20 females) and in 60 patients with acute leukosis (27 males and 33 females), with chronic myeloleukosis - 23 (10 males and 13 females), and 25 patients with chronic lympholeukosis (II males and 14 females). Another 15 patients (7 males and 8 females) were studied at the initial stage, recurrence, recidivation of acute leukosis (AL). DALA excreted with the urine, manifested no significant discrepancy as compared with the controls of the three groups examined patients and in all the three stage of AL, whereas PBG was moderately reduced in the AL patients - initial stage and recurrence and was within the normal limits in the patients with chronic myeloleukosis (CML) and chronic lympholeukosis (CLL). The urine excreted coproporhyrin was increased, to various degrees, in the patients with CML and CLL and at the initial stage, recurrence and recidivation of AL, whereas uroporhyrin was within the norm. Coproporphyrin, excreted in the feces, was increased in all three groups of patients with leukosis and in the three stages of AL, whereas the other fractions showed no significant difference as compared with the controls. It could be concluded, from the results obtained, that porphyrins metabolism is disturbed in the patients with leukosis.

A simple solid phase radioimmunoassay specific for human C3b to detect C3b receptors on human lymphoblastoid cell surfaces.

Our aim was to detect C3b receptors on human lymphoblastoid cells using a solid phase radioimmunoassay (RIA) specific for human C3b. RIA was performed by coupling rabbit antihuman C3b to acrylamide beads to make immunobeads. The specificity and sensitivity of binding of 125I-C3b to immunobeads allowed the detection of as little as 6 X 10(-10) M unlabeled human C3b. The cells were incubated with a C3b concentration (10(-9) M) giving 25% inhibition in the RIA. The concentration of unbound C3b was then measured in the cell supernatants using RIA. Results showed that: (a) loss of C3b antigen in the cell supernatant was not due to degradation of C3b molecules, (b) C3b binding could be detected at 37 degrees C on the four B cell lines, but not on the two T cell lines or on the two non T-non B cell lines tested, (c) C3b bound on the B lymphoblastoid cells was not cleaved, neither into iC3b nor C3c and C3d fragments, supporting the presence of C3b receptors on the cells tested. This method allows screening of a variety of C3b receptor-positive cells.

Cytoplasmic RNA from normal and malignant human cells shows homology to the DNAs of Epstein-Barr virus and human adenoviruses.

Cytoplasmic RNA prepared from several human cell lines and tissues was hybridised to DNA from Epstein-Barr virus, human adenovirus types 2, 3 and 12 and human papovaviruses BK and JC. RNA from all the cells, regardless of whether or not they were virally infected, hybridised to specific regions of the Epstein-Barr virus or adenovirus genomes but not to papovavirus DNA. The cellular cross-hybridising species appear to be repetitive sequences which are conserved in higher eukaryotes. Mismatch estimations indicate a high degree of homology between the viral and host sequences. Detailed analysis of selected regions of viral DNA failed to reveal any primary-structural peculiarities.

The analysis with monoclonal antibodies of the heterogeneity of Ia glycoproteins on chronic lymphocytic leukemia cells.

The accessible Ia molecules on the surface of chronic lymphocytic leukemia (CLL) cells were quantitated in the cellular radioimmunoassay with saturating concentrations of monoclonal antibodies. Monoclonal antibody 21w4, like DA/2 antibody, recognizes monomorphic determinants of human Ia antigens. The amount of 21w4 or DA/2 bound to CLL cells derived from eight patients (varying from 2.6 to 13.9 x 10(5) molecules/cell) appears to be the maximum observed with the antibodies studied. Two other antibodies, 18d5 and 21r5, although also directed at nonpolymorphic Ia determinants, bind differentially to CLL cells, with the ratios of 21r5/21w4 and 18d5/21w4 varying from 0.08 to 0.90. Sequential immmunoprecipitation studies have established that the four epitopes 18d5, 21r5, 21w4, and DA/2 were present on the same molecules. All Ia molecules express 21w4 and DA/2 epitopes, whereas only certain subsets of Ia molecules carry accessible 21r5 or 18d5 epitopes. Competitive binding studies showed that the epitopes recognized by the four monoclonal antibodies were different. Monoclonal antibodies 21r5 and 21w4 did not inhibit each other's binding. Furthermore, binding of 21w4 to CLL cells potentiated the binding of 125I-21r5 IgG to the same cells, suggesting that binding of 21w4 antibody induces a conformational change in the molecule that renders 21r5 epitopes more accessible.

Vindesine receptors in cells of a human leukaemia cell line.

To determine whether vindesine receptors are present in human leukaemic cells, K562 cells (established from chronic myelogenous leukaemia in blastic crisis) were incubated with 3H-vindesine. Binding of 3H-vindesine increased with incubation time and with increase in number of K562 cells. However, when excessive amounts of nonradioactive vindesine were added, the 3H-vindesine was displaced. Binding of 3H-vindesine was only inhibited by vinblastine, vincristine and vindesine. These results suggest that K562 cells have receptors for vindesine and that these receptors are common to vinca alkaloids. Scatchard analysis showed that the number of vindesine receptors differed according to the kind of cells tested. K562 and a T-cell leukaemia-derived cell line, MOLT-4, had more receptors than an acute promyelocytic leukaemia-derived cell line, HL-60, and normal blood lymphocytes. The degree of vindesine affinity to receptors did not differ markedly among the above-mentioned cells.

Dimer-size DNA circles in a leukemic cell immortalized with the Epstein-Barr virus.

The intracellular state of the 30 viral genome equivalents of Epstein-Barr virus (EBV) DNA carried in latent form by the CII cell line, established from a chronic lymphocytic leukemia patient, has been partially characterized. The CII line, which has markers confirming its tumor origin, extends the analysis of the intracellular state of EBV DNA to include other, non-Burkitt lymphoid tumor cells. Monomer-size, free, circular EBV genomes, the major intracellular viral DNA species in other EBV-transformed cells, were absent or present in only minor amounts. Instead, EBV DNA sequences were found associated with a circular DNA form twice the size of the 110 x 10(6) Mr EBV genome. Though circular dimers of mtDNA have been found exclusively in human leukemic lymphocytes, the CII line is similar to normal cells in having only monomer-size mtDNA molecules, which can occur either singly or as catenated forms of two or more interlocking 5-micrometer mtDNA circles.

[Microspectrophotometric method of studying the nuclear DNA of lymphoid cells in the lymph nodes in systemic blood diseases]. / Mikrospektrofotometricheskii metod issledovaniia iadernoi DNK limfoidnykh kletok limfaticheskikh uzlov pri sistemnykh zabolevaniiakh krovi.

Basing on studies into nuclear DNA of lymphoid cells in histological sections of lymph nodes from patients with malignant non-Hodgkin's lymphoma, chronic lymphatic leukemia and myeloma, a modified scanning integrating digital microspectrophotometer enables one to differentiate between these diseases in terms of the content of nuclear DNA in lymphoid cells. The minimal content of nuclear DNA was found in malignant non-Hodgkin's lymphoma, the maximal during myeloma, with this content ranking intermediate in chronic lymphatic leukemia. The data obtained might be used as additional criteria in diagnosing the diseases in question.

Glucocorticoid receptors, clinical characteristics, and implications for prognosis in chronic lymphocytic leukemia.

The cytoplasmic glucocorticoid-receptor (GR) content was analyzed in peripheral leukocytes from 52 nonselected patients with chronic lymphocytic leukemia (CLL). Forty-six patients had measurable GR levels. Six patients lacking measurable GR and seven of eight patients with a low GR content had inactive disease. However, there was no significant difference in average GR content between patients with progressive or nonprogressive disease. Twelve GR-positive patients were treated with prednisolone as the sole medication. Seven patients responded, some in several treatment sessions. There was no correlation between GR-content and clinical response. Thus, in our experience, cytoplasmic GR measurement has only limited predictive value.

Monoclonal immunoglobulin of CLL lymphocytes detected by radioimmunofixation with 125I-protein A of Triton X-100 solubilized lymphocyte material.

A sensitive radioimmunofixation method (detection limit 1 mg/1 for monoclonal IgM) utilizing 125I Protein A and immunofixation for demonstration of monoclonal immunoglobulin in Triton X-100 extracts of isolated chronic lymphocytic leukemia (CLL) lymphocytes is presented. Thirty-two untreated patients with clinically typical CLL were studied and the results compared to those obtained by immunofluorescence. Monoclonal IgM was detected with similar frequency by the two methods (69% and 75%), but IgD was detected less frequently by radioimmunofixation (25% and 40%). When the results obtained with the two methods were combined, all of the cases were immunoglobulin positive. Two complete IgM type M-components were demonstrated in two cases, an excess of seemingly free heavy chains was found in four cases, and an excess of seemingly free light chains in six cases.

Tubulin as a major cell surface protein in human lymphoid cells of leukemic origin.

Surface-exposed proteins of vinblastine-sensitive human lymphoid cell line of leukemic origin (CCRF-CEM) were examined by the lactoperoxidase-catalyzed iodination and two-dimensional polyacrylamide gel electrophoresis methods. Spots which comigrate with bovine brain tubulin and rabbit muscle actin were prominently labeled in the whole membrane but not in the high-speed supernatant fraction of the disrupted cells. Mild trypsinization of labeled cells removed the iodinated tubulin and actin without significantly affecting the protein staining pattern. Iodination of normal human lymphocytes resulted in no labeling of the tubulin or actin. The presence of surface-exposed tubulin in this leukemic cell line suggests a possible mechanism for their enhanced sensitivity to the cytotoxic action of vinblastine.

Human T-cell malignancies: Correlative clinical, histopathologic, immunologic, and cytochemical analysis of 23 cases.

Twenty-three T-cell neoplasms were investigated for their reactivity with the OKT monoclonal antibodies and expression of certain cytochemical markers. Fourteen neoplasms with diverse histopathologic features, T-cell chronic lymphocytic leukemia, mycosis fungoides, the Sézary syndrome, T-immunoblastic sarcoma, and a pleomorphic large-cell lymphoma, expressed the T helper cell phenotype, OKT3+T4+. Nine other neoplasms displayed marked inter- and intra- tumor heterogeneity. Seven of these cases, lymphoblastic lymphoma, T-cell acute lymphoblastic leukemia, and tumors with feature of T-immunoblastic sarcoma or the multilobated lymphoma of Pinkus, expressed intrathymic phenotypes. The other 2 cases, a lymphoblastic lymphoma and a so-called Lennert's lymphoma, expressed the previously undescribed OKT3+T10+ phenotype. These studies demonstrate that the T-cell malignancies are divisible into phenotypes corresponding to normal maturational stages of T-cell differentiation and functionally distinct T-cell subsets. Such studies should provide a basis for understanding the biologic heterogeneity, clinical diversity, and significance of the variable cytomorphologic characteristics of T-cell malignant tumors and assist in the further delineation of normal human T-cell heterogeneity.

Chronic lymphocytic leukemia: demonstration of cell surface structural heterogeneity by lectin affinity chromatography of cell extracts.

I have used lectin affinity chromatography of radiolabelled components in cell extracts to examine molecular heterogeneity of membrane glycoproteins from 10 cases of chronic lymphocytic leukemia (CLL). Different complements of radiolabelled surface glycoproteins were found in extracts from cells with different lectin binding characteristics. Additional confirmation of heterogeneity was obtained by analyses of hexose and sialic acid contents of the extracts and various lectin-adherent fractions. The fact that CLL cells from different patients may carry different cell surface oligosaccharides provides the possibility that clinical variability in this disorder may relate in part to this molecular heterogeneity. The data further indicate that different lectins may bind to different saccharide structures at CLL cell surfaces. CLL cells represent excellent source material for isolation and purification of specific lectin receptor molecules.