Altered DNA topoisomerase II activity in Chinese hamster cells resistant to topoisomerase II inhibitors.

Most DNA intercalators and epipodophyllotoxins inhibit mammalian topoisomerase II by trapping the enzyme within DNA cleavage complexes that can be detected in cells as protein-associated DNA strand breaks. We have characterized previously a line of Chinese hamster cells (DC3F/9-OHE cells) the resistance of which to the cytotoxic effect of intercalators and etoposide is associated with a reduced formation of protein-associated DNA strand breaks. In the present study, topoisomerases of these cells were compared to those of the parental sensitive cells (DC3F). NaCl extracts (0.35 M) of isolated DC3F/9-OHE nuclei did not form 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced DNA-protein linking, whereas DC3F nuclear extracts did. In addition, DC3F/9-OHE nuclear extract had an unusually high level of DNA linking activity in the absence of 4'-(9-acridinylamino)methanesulfon-m-anisidide. Topoisomerases II from DC3F/9-OHE and DC3F nuclei appeared similar qualitatively. DC3F/9-OHE nuclear extract had approximately twice less topoisomerase II molecules than did DC3F nuclear extract but similar topoisomerase II activity. Topoisomerase I activities appeared also similar in sensitive and resistant cells. However, part of DC3F/9-OHE topoisomerase I copurified with a DNA linking activity which was not present in DC3F nuclei. This unusual DNA linking activity was not sensitive to the stimulatory effect of 4'-(9-acridinylamino)methanesulfon-m-anisidide.

Structural characterization of CD6: properties of two distinct epitopes involved in T cell activation.

Studies from our laboratory have shown that anti-T12, a mAb which recognizes CD6, is a macrophage-dependent mitogen for human T cells and can augment T cell autoreactivity in vitro. To obtain additional information regarding the potential biological role of CD6 we sought to further characterize its biochemical properties. The CD6 molecule on 125I-surface-labeled T cells and by Western blot analysis was a monomer of mol. wt 130,000 under reducing conditions and mol. wt 117,000 under non-reducing conditions, suggesting the presence of intrachain disulfide bonds. The polypeptide contains a protease sensitive site. In activated T cells, the protein was serine phosphorylated. Analysis of biosynthetically labeled CD6 in the presence of tunicamycin revealed a reduction in mol. wt from 130,000 to 100,000, indicating that the polypeptide is extensively N-glycosylated. The mAb, anti-2H1, had been shown to activate T cells in combination with PMA or the anti-T11(3) mAb but, unlike anti-T12, not in the presence of macrophages alone. The present studies demonstrate by sequential immunoprecipitation that these two mAbs recognize the same polypeptide. However, Western blot analysis and indirect immunofluorescence cross-blocking studies demonstrate that the two mAbs recognize different determinants on CD6. Anti-T12 recognizes an epitope that is present only under non-reducing/non-denaturing conditions, while anti-2H1 recognizes an epitope that is also preserved under reducing/denaturing conditions. A direct comparison of activation properties of the mAbs confirmed that anti-T12 was optimally mitogenic in the presence of macrophages but not PMA, while, conversely, anti-2H1 was optimally mitogenic in combination with PMA but not macrophages, suggesting that the differences in epitope specificity may account for the distinct activation properties of each mAb. Taken together, the structural and functional data strongly suggest that the CD6 membrane glycoprotein may function as a physiologically important receptor structure on human T lymphocytes.

Improved conditions for activity gel analysis of DNA polymerase catalytic polypeptides.

In a study of mouse DNA polymerase catalytic polypeptides using activity gel analysis, it was found that the sensitivity of detection of purified enzymes is markedly increased by addition of a heterogeneous mixture of proteins to the enzyme sample prior to electrophoresis (Karawya E., and Wilson, S.H. (1982) J. Biol. Chem. 257, 13,129-13,134). This modification and the use of a micromolar level of [32P]dNTP substrate are the basis of an improved activity gel assay for DNA polymerase catalytic polypeptides. This modified assay is several orders of magnitude more sensitive than the original procedure (Spanos, A., Sedgwick, S.G., Yarranton, G.T., Hubscher, U., and Banks, G.R. (1981) Nucl. Acids Res. 9, 1825-1839), and it enables measurement of two reference enzymes, calf beta-polymerase and Escherichia coli DNA polymerase I large fragment, in the picogram range. Further, it was found that it is essential to survey different lots of sodium dodecyl sulfate to identify those which enable high enzyme activity signals after renaturation.

Activation of in vitro proliferation of human T cells by a synthetic peptide of toxic shock syndrome toxin 1.

A 21-mer synthetic peptide (KGEKVDLNTKRTKKSQHTSEG), designated TSST-1(58-78), was constructed from the primary structure of the toxic shock syndrome toxin 1 (TSST-1). The peptide reacted with a panel of neutralizing monoclonal antibodies (MAbs) to whole TSST-1 in solid-phase immunoassays. TSST-1(58-78) promoted the in vitro proliferation of human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Minimum dose required for stimulation (P less than or equal to .05 microM) was 0.75 microM peptide. This mitogenic effect was abrogated by incubation of the peptide with MAbs to whole TSST-1 before addition to PBMC. The ability of TSST-1(58-78) to stimulate the proliferation of highly purified resting human T cells was analyzed. Significant proliferation (P less than or equal to .01) was observed only in the presence of increasing populations of monocytes added to the cultures. Adherent human monocytes exposed to TSST-1(58-78) released tumor necrosis factor. Thus, some of the immunoregulatory properties attributed to TSST-1 are demonstrated by the region of the toxin represented by the peptide TSST-1(58-78).

Anti-T12, an anti-CD6 monoclonal antibody, can activate human T lymphocytes.

Anti-T12 is a murine IgM mAb that recognizes the 130-kDa CD6 glycoprotein on mature human T lymphocytes. Examination of the in vitro effects of this mAb on freshly isolated T cells demonstrates that anti-T12 can induce T cell activation. Such activation is macrophage-dependent, and optimal stimulation occurs with 0.2 to 5 micrograms/ml of purified mAb. This response to soluble mAb is detectable at day 4 to 5 of culture, and peak [3H]TdR uptake is observed at day 7. In a highly similar fashion, the mAb causes a marked augmentation of the autologous mixed lymphocyte reaction, without altering the kinetics of that response. Although optimal anti-CD3 mAb induced mitogenesis is unaffected by anti-T12, suboptimal stimulation of macrophage-depleted T cells by small amounts of immobilized anti-CD3 can be dramatically enhanced when anti-CD3 and anti-T12 are cross-linked together. A soluble nonmitogenic anti-CD3 mAb completely inhibits anti-T12-mediated T cell activation. IL-2R expression is induced by anti-T12 stimulation in 10 to 30% of T cells, and T cell proliferation is substantially inhibited by anti-IL-2R mAb, indicating that anti-T12 induced T cell activation and proliferation utilizes an IL-2-dependent pathway. Isolated CD4+ but not CD8+ cells are stimulated to proliferate, but the CD8+ cells in unseparated T cell preparations activated by anti-T12 do proliferate comparably to the CD4+ cells in such cultures. These data indicate that relatively weak activation of T cells via the TCR/CD3 complex may be augmented significantly by CD6-mediated signals induced by the anti-T12 mAb. The findings suggest that the CD6 T cell membrane protein may have the capacity to function as a physiologically important structure involved in the regulation of T cell activation.

Studies on the mechanism of DNA polymerase alpha. Nascent chain elongation, steady state kinetics, and the initiation phase of DNA synthesis.

A detailed study of the mechanism of nascent chain elongation and of steady state kinetics of purified mouse DNA polymerase alpha has been conducted. Polymerization was examined using a model replication system of poly(dT) as template, oligo(rA) as primer, and dATP as nucleotide substrate, and the probability of chain termination was determined by measurement of the precise chainlength of the products. Reactions were conducted under conditions where products were not utilized as primer. Product chainlength analysis indicated that alpha-polymerase acted in a processive fashion, elongating the primer by the stepwise addition of up to 20 dAMP residues before dissociating. The probability of termination after each dAMP addition depended upon the chainlength of the product and upon the presence of several agents; spermine, spermidine, putrescine, nalidixic acid, or PPi caused a marked increase in termination after the first dAMP addition, and conversely, mouse helix destabilizing protein-1 caused the enzyme to continue extending the same product chain until 18 to approximately 35 dAMP residues had been added. From these and other data, it is concluded that the kinetic mechanisms of termination after the first dAMP addition and after subsequent dAMP additions are different. With this information on how alpha-polymerase elongates a nascent primer(dA)n molecule, a kinetic model and appropriate steady state rate equations were obtained for analysis of substrate initial velocity data and termination probabilities. The substrate kinetic patterns and PPi product inhibition results were consistent with the ordered Ter Ter mechanism Bi Uni Uni Bi Ping Pong proposed in the model, and the model also permits a rational explanation for the differences in termination probability and for the fact that substrate initial velocity plots were linear even though multiple residues of dATP combined with the enzyme during each catalytic cycle. In addition, the results suggest that a rate-limiting step in the steady state occurs at the transition between initiation and elongation, and that higher levels of template.primer increase the rate of this step. This secondary effect of template.primer is discussed in relation to other polymer-forming enzymes, and various kinetic mechanisms which require the presence of two template.primer-binding sites, effector and catalytic, are discussed for their fit to the experimental data.

Properties and applications of new monoclonal antibodies raised against calf DNA polymerase alpha.

A panel of 12 hybridoma cell lines secreting monoclonal antibodies against alpha-polymerase were prepared by fusion of mouse myeloma cells and spleen cells of a rat immunized with homogeneous calf thymus alpha-polymerase. Hybridomas were selected and cloned on the basis of immunobinding to pure alpha-polymerase in solid phase radioimmunoassay. Antibodies secreted by these cells eventually were purified in milligram quantities from ascites fluids. These antibodies, all of the rat immunoglobulin M class, cross-reacted with alpha-polymerases from calf and monkey cells as revealed by immunobinding in radioimmunoassay and by immunoprecipitation of DNA polymerase activity. The antibodies were not capable of neutralizing the enzyme activity. With the methods described these antibodies may be used to immunoprecipitate alpha-polymerase from crude extracts of mammalian cells and to measure levels of the enzyme protein.

P1 plasmid replication: measurement of initiator protein concentration in vivo.

To study the functions of the mini-P1 replication initiation protein RepA quantitatively, we have developed a method to measure RepA concentration by using immunoblotting. In vivo, there are about 20 RepA dimers per unit-copy plasmid DNA. RepA was deduced to be a dimer from gel filtration of the purified protein. Since there are 14 binding sites of the protein per replicon, the physiological concentration of the protein appears to be sufficiently low to be a rate-limiting factor for replication. Autoregulation is apparently responsible for the low protein level; at the physiological concentration of the protein, the repA promoter retains only 0.1% of its full activity as determined by gene fusions to lacZ. When the concentration is further decreased by a factor of 3 or increased by a factor of 40, replication is no longer detectable.

Biosynthesis and post-translational modification of CD6, a T cell signal-transducing molecule.

CD6 (T12) is a 130-kDa glycoprotein present on the surface of human T cells. Previously, we demonstrated that the anti-T12 and anti-2H1 monoclonal antibodies recognized different epitopes on CD6, and both were capable of transducing activation signals to T cells. Anti-T12 augmented suboptimal signaling via the TCR/CD3 complex and directly activated separated CD4+ but not CD8+ cells. Structural characterization of CD6 revealed that it contained intrachain disulfide bonds, was N-glycosylated, and in activated cells was phosphorylated on serine. Given the functional significance of CD6 and its involvement in signaling via CD3 and CD2 pathways, we examined in detail the biosynthesis, structural characteristics, and phosphorylation properties of this receptor-like molecule. These studies demonstrate that the nascent CD6 polypeptide on both T cells and thymocytes in 88 kDa, and the immature N-glycosylated form is 110 kDa. After maturation of N-linked glycan and addition of sulfated O-linked oligosaccharide, CD6 appears on the cell surface as a molecule of 130 kDa. CD6 is phosphorylated in resting cells and can be hyperphosphorylated when stimulated by phorbol 12-myristate 13-acetate, indicating that it may participate in the major common signaling pathway mediated through protein kinase C. Concanavalin A-activated cells are phosphorylated at an additional site(s) on the molecule and cannot be hyperphosphorylated with phorbol 12-myristate 13-acetate. These physical features reveal additional clues about the physiological role of CD6 and its mechanism of signal transduction and strongly suggest that CD6 represents a physiologically important membrane receptor involved in T cell activation.