Myc oncogene activation in B and T lymphoid tumours

The chromosome translocations characteristic of certain B lymphoid tumours associate the myc oncogene and immunoglobulin loci. The typical t(12;15) in murine plasmacytomas and analogous t(14;8) in Burkitt lymphomas couple the myc coding region to one of the switch recombination regions within the immunoglobulin heavy (H) chain locus; hence the switch machinery may promote some translocations. Significantly, translocation induces constitutive myc expression, the untranslocated myc allele remaining silent. The predilection for breakpoints near the 5' end of the c-myc gene may reflect selection for altered myc regulation. In most tumours, the stimulatory effect of the H locus context is not understood, but an H locus enhancer participates in some tumours, including one displaying a novel transposition. The variant (6;15) translocations found in about 15% of plasmacytomas involve the myc band and the region of chromosome 6 where the kappa locus lies. The t(6;15) is shown here to represent an exchange between C kappa and a chromosome 15 locus (designated pvt-1) which lies unexpectedly far from c-myc. The association of myc expression with pvt-1 alterations suggest that myc can be activated at a distance. Myc has also been implicated in some T lymphomas by detection of proviral inserts near myc and also, surprisingly, within the pvt-1 locus. Inserts near myc appear to activate its expression via the retroviral enhancer.     

Studies directed toward testing the "channeling" hypothesis--ribonucleotides----DNA in leukemia L1210 cells

Experiments were carried out to test for the presence of "channeling" in L1210 cells. L1210 cells were incubated in culture in the presence of labeled cytidine and "cold" deoxycytidine and conversely, in the presence of labeled deoxycytidine and "cold" cytidine. Cytidine did not inhibit the incorporation of [14C]deoxycytidine into DNA while deoxycytidine decreased the incorporation of [14C]cytidine into DNA. Further, in L1210 cells there was not a coordinate inhibition of thymidylate synthetase when either DNA polymerase was inhibited (aphidicolin) or ribonucleotide reductase was inhibited (hydroxyurea). These data indicate that leukemia L1210 cells do not selectively channel ribonucleotides to DNA through a tightly coupled enzyme complex.     

Localization of constitutive heat shock proteins in developing ascidians

Heat shock proteins (HSP) are a group of highly conserved proteins that regulate protein folding and ameliorate the effects of environmental stress. In the present study, the question of whether or not ascidian oocytes, embryos and larvae constitutively synthesize HSP was studied using HSP 60 and HSP 70 antibodies. Developmental stages obtained from Boltenia villosa, Cnemidocarpa finmarkiensis, Styela montereyensis and Corella willmeriana were examined for HSP using indirect immunocytochemistry. Myoplasm in oocytes and unfertilized eggs reacted with HSP 60 and 70 antibodies. HSP signals dramatically moved into the vegetal egg cytoplasm during ooplasmic segregation and colocalized with the myoplasm. In cleavage-stage embryos, HSP signals were partitioned with the myoplasm into muscle progenitor blastomeres and HSP signals were evident in the tail muscle cells of larvae. Immunoblots of proteins extracted from oocytes, eggs, embryos and larvae indicate that anti-HSP 60 recognizes a single band having an estimated molecular weight of 60 kDa. Egg centrifugation experiments suggest that most of the ascidian myoplasmic HSP are mitochondrial proteins. These results raise an intriguing possibility that mitochondria associated with the myoplasm perform biochemical functions that are unique to the embryonic muscle cell lineage.     

Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.

Activation of the cell surface receptor Fas/APO-1 (CD95) induces apoptosis in lymphocytes and regulates immune responses. The cytoplasmic membrane protein Bcl-2 inhibits lymphocyte killing by diverse cytotoxic agents, but we found it provided little protection against Fas/APO-1-transduced apoptosis in B lymphoid cell lines, thymocytes and activated T cells. In contrast, the cowpox virus protease inhibitor CrmA blocked Fas/APO-1-transduced apoptosis, but did not affect cell death induced by gamma-radiation or serum deprivation. Signalling through Fas/APO-1 did not down-regulate Bcl-2 or induce its antagonists Bax and Bcl-xS. In Fas/APO-1-deficient lpr mice, Bcl-2 transgenes markedly augmented the survival of antigen-activated T cells and the abnormal accumulation of lymphocytes (although they did not interfere with deletion of auto-reactive cells in the thymus). These data raise the possibility that Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.     

The molecular weight of Ehrlich tumor cell ribonucleotide reductase and its subunits: Effector-induced changes

The molecular weights of Ehrlich tumor cell ribonucleotide reductase and its individual components were determined by sedimentation equilibrium in the Beckman Airfuge. The distribution of enzyme after sedimentation equilibrium was determined by measurement of the CDP reductase and ADP reductase activities associated with ribonucleotide reductase. The apparent molecular weight of the intact enzyme was 304,000 when assayed for CDP reductase and 254,000 when assayed for ADP reductase. This difference in apparent molecular weights was statistically significant with a P value of 0.0002. The molecular weights of the individual components of ribonucleotide reductase were determined in a similar fashion by assaying in the presence of an excess of the complementary component. The non-heme iron component had a molecular weight of 81,000 when assayed for either CDP or ADP reductase activity. The effector-binding component had an apparent molecular weight of 127,000 when assayed for CDP reductase and 95,000 when assayed for ADP reductase. This difference in apparent molecular weights was statistically significant with a P value of 0.004. The effectors ATP and dGTP altered the apparent molecular weights of the intact enzyme and individual components. In the presence of ATP the molecular weight of intact CDP reductase was 481,000 while the apparent molecular weight of the effector-binding component of CDP reductase alone was 418,000. In the presence of dGTP, the molecular weight of intact ADP reductase was 293,000 while the apparent molecular weight of the effector-binding component of ADP reductase alone was 154,000. These results indicate that the proportion of the non-heme iron component and the effector-binding component is not equimolar and that the composition of the enzyme is not constant but is altered by the presence of effectors. Our data also suggest that CDP reduction and ADP reduction are catalyzed by different molecular species of the enzyme which apparently have different effector-binding components.