Human homologue of murine T200 glycoprotein.

We report the identification of the human homologue of murine T200 glycoprotein. Peptide-mapping experiments suggest that the structure of the glycoprotein is highly conserved between the two species. Many of the properties of human T200 homologue are similar to those of murine T200 glycoprotein: it is broadly distributed within the hematopoietic system but is not detectable on nonhematopoietic cells; there are also structural differences between the forms of the glycoprotein found on T and B lymphoblastoid cell lines. These results suggest the homologous glycoproteins may play similar functional roles in both species.

High resolution proton magnetic resonance spectral characteristics of water, lipid, and protein signals from three mouse cell populations.

The high resolution proton magnetic resonance spectral properties of three C57BL/6J mouse cell populations (EL4 ascites tumor cells, normal spleen leukocytes, and normal erythrocytes) were investigated. Packed cell pellets were investigated at 100 MHz and 13.56 MHz. The 100-MHz spectra contained identifiable non-water proton resonances as well as the water resonance. The major non-water resonances from the EL 4 cells and normal leukocytes resembled resonances from lipid extracts, whereas the non-water resonances from erythrocytes resembled resonances from hemoglobin solutions. The reciprocals of the water proton spin-lattice relaxation times were roughly proportional to the total sample water content at both 100 and 13.56 MHz. The estimated slopes of these plots were frequency-dependent. The water proton relaxation rate generally increased with increasing total protein content of the packed cell pellets.

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.

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.

Chromatin phospholipids in normal and chronic lymphocytic leukemia lymphocytes.

Certain phospholipids are associated with the nonhistone chromosomal proteins extracted from normal B- and chronic lymphocytic leukemia lymphocytes. The ratio of phospholipids to nonhistone chromosomal proteins was constant with the different methods used for isolating nuclei and extracting the chromatin, although the various methods allowed a different recovery of total lipids from chromatin. Three phospholipids were extractable from the nonhistone protein fraction, but their respective ratios varied in chronic lymphocytic leukemia compared to normal B-lymphocytes. The most significant variation concerns the reduction of sphingomyelin content in leukemic lymphocytes, since this prospholipid in vitro affects both DNA stability and transcription.

Glucocorticoid receptors in adult acute lymphoblastic leukemia.

Glucocorticoid receptors were studied in leukemic cells from 14 newly diagnosed adults with acute lymphoblastic leukemia. Receptors were found in all cases; total receptor levels ranged from 1,348 to 18,697 sites per cel (median, 7,553). To determine the utility of glucocorticoid receptor levels for predicting response to glucocorticoid therapy, lymphoblasts were studied for receptors on nine occasions, and the patients were then treated with dexamethasone as a single agent. Response to glucocorticoid therapy appeared to correlate with lymphoblast receptor level. Five of six patients with greater than 4,500 total receptor sites per cell responded; all three patients with less than 4,500 receptors failed to respond. Glucocorticoid receptor level did not correlate with response to combination chemotherapy, duration of remission, or survival. Glucocorticoid receptors were studied at the initial presentation and following relapse in five patients. In two patients, the receptor level did not change following therapy; both patients at diagnosis had leukemias resistant to glucocorticoids. Three patients at relapse developed leukemias with lower receptor levels; these patients had leukemias sensitive to glucocorticoids at diagnosis. Our data suggest that the glucocorticoid receptor levels of lymphoblasts may be useful in predicting response to glucocorticoid therapy in adult acute lymphoblastic leukemia. They also suggest that, following relapse, cases initially sensitive to glucocorticoid may develop resistance by selection of cells with fewer receptors.

Properties of chromosomal proteins of human leukemic cells.

Purified chromatin isolated from lymphocytic cells derived from patients with acute leukemia, or other lymphoproliferative disorders has been compared with chromatin isolated from normal human lymphocytic cells by gel electrophoresis and differential gradient ultracentrifugation. Thermal denaturation studies showed higher Tm values for chromatin from leukemic cells, as compared to that of lymphocytic cells from normal donors or patients with infectious mononucleosis, reflecting the diverse complexity of these chromatins with respect to their varying chemical compositions. There are significant differences in the ratios of DNA:RNA:protein, as well as in the ratios of chromatin-associated histone and non-histone proteins; although chromatin-associated histones were more homogeneous than were the non-histone proteins, as adjudged by amino acid analyses and acrylamide gel electrophoresis. These differences in chromatin structure may relate to the differences in gene expression characteristic of these lymphocytic cells. The chromosomal acidic proteins isolated from the purified chromatin of human leukemic cells greatly stimulated the template activity of the chromatin in in vitro RNA synthesis. The non-histone proteins selectively interact with chromatins and influence the RNA polymerase reactions, indicating that there is selective tissue specificity of non-histone proteins.

Methods of quantitative autoradiography using incident light microphotometry.

Several different approaches of automated grain counting of microautoradiographic grain densities have been reported in the literature. Application of grain counters to cell biology is limited, however, primarily due to shortage of methods allowing the interpretation of grain counts on a molecular basis. Two suitable methods of quantitative autoradiography at the cellular level are reviewed, developed for the isotopes 14C and 125 I. They permit evaluation of absolute radioactivity in autoradiographs and, thus, determination of synthesis processes such as deoxyribonucleic acid synthesis, and of antigen densities on cell surfaces. In this approach towards quantitative autoradiography, grain densities are compared photometrically over labeled cells and over a standard source on the same autoradiograph. Allowance has to be made for the specific geometric factors of the isotope used. This can be advantageously done with an integrating type of measurement using incident light bright-field. With this type of recording, there is an exponential dependence of the photometric values on the radioactive dose. As an example of application, results are presented of the deoxyribonucleic acid synthesis rate of human myelocytes in aplastic anemia and of the immunoglobulin G density on lymphocyte membranes in the normal state and in chronic lymphocytic leukemia.

Double stranded RNA in heterogeneous nuclear RNA from normal and chronic lymphocytic leukemic lymphocytes.

Tritium labelled heterogeneous nuclear RNA (HnRNA) from normal and chronic lymphocytic leukemic (CLL) lymphocytes was investigated before and after fractionation into non-poly(A) containing (-HnRNA) and poly(A) containing (+HnRNA) HnRNA with respect to double stranded RNA (dsRNA). Statistically significant higher amounts of rapidly labelled RNA were recovered from CLL lymphocytes when compared to normal cases. Within the CLL cases a significant linear correlation (r = 0.95) was found between white blood cell counts and the amount of dsRNA in total HnRNA. After fractionation into (-) and (+) HnRNAs the ratios of dsRNAs, expressed as the dsRNA in (-) HnRNA divided by the dsRNA in (+) HnRNA, was lower than the corresponding values in normal cases for all the CLL cases except one. The relationship between (+) HnRNA and the total dsRNA level was different when comparing CLL and normal lymphocytes indicating a RNA processing abnormality.

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.

Periodic acid-Schiff reaction and prognosis in lymphoblastic leukaemia.

Diagnostic bone marrow smears from 132 patients with acute lymphoblastic leukaemia, (ALL) were stained simultaneously by the periodic acid-Schiff (PAS) reaction, and the blast cell positivity was assessed quantitatively. The patients fell naturally into two unequal groups: those with more than 20% PAS-positive blast cells (44 patients) and those with less (88 patients). There was no relation between the degree of positivity and age, sex, or presenting leucocyte count. Actuarial survival studies showed that the group with more than 20% PAS-positive blast cells survived longer, but that this difference assumed statistical significance only after the exclusion of patients over 14 years old and those with high white cell counts at the time of diagnosis. It appears that the PAS reaction can identify long survivors among patients with ALL, but not in the absence of features strongly associated with a poor prognosis.

[Intragenomic specificity of DNA methylation in animals. Qualitative differences in tissues and changes in methylation of repeating sequences during aging, carcinogenesis and hormonal induction]. / Vnutrigenomnaia spetsifichnost' metilirovaniia DNK u zhivotnykh. Tkanevaia raznokachestvennost' i izmenenie metilirovaniia povtoriaiushchikhsia posledovatel'nostei pri starenii, kantserogeneze i gormonal'noi induktsii.

5-methylcytosine (m5C) is nonrandomly distributed in mammalian genome. The unique sequences in DNA of all species studied (mouse, rat, cow) methylated to a similar extent and are characterized by a minimal m5C content (0.8--0.9 mole%). The very renaturating sequences (including satellite DNA) possess a maximal m5C amount. The rarely repeated (10 to 1000 folds) sequences which are known to contain genes for rRNA and tRNA as well as for histones are characterized by an elevated level of methylation. It is established that tissue differences in m5C content in DNA as well as decrease in the DNA methylation level with age and on spontaneous lympholeukosis in cattle and, on the contrary, increase in DNA methylation degree in rat liver as a result of induction with hydrocortisone, are due to tissue specific changes in the methylation level of repeated but not unique sequences. The methylation level of sequences differing in the reiteration degree seems to be mainly due to CG-dublet frequencies. In the unique sequences this particular dinucleotide is almost completely methylated. Nevertheless, the multiplicity of DNA methylases in the animal nucleus is not ruled out. Methylation of the CG sequence is supposed to protect animal DNA's, especially structural genes, against restriction endonucleases. The decrease in DNA methylation in animals with age and on lympholeukosis detected by us is considered to be one of the possible reasons for chromosome lesions and for distortions in DNA replication and transcription.