Leo Sachs

The induction of clones of normal mast cells by a substance from conditioned medium

Abstract A substance in conditioned medium from unirradiated and x-irradiated embryo cell feeder layers can induce the multiplication of colony forming cells and the formation of mast cell clones from single cells. The substance has a high degree of thermostability and is not dialyzable.

NQO1 stabilizes p53 through a distinct pathway

Wild-type p53 is a tumor-suppressor gene that encodes a short-lived protein that, upon accumulation, induces growth arrest or apoptosis. Accumulation of p53 occurs mainly by posttranslational events that inhibit its proteosomal degradation. We have reported previously that inhibition of NAD(P)H: quinone oxidoreductase 1 (NQO1) activity by dicoumarol induces degradation of p53, indicating that NQO1 plays a role in p53 stabilization. We now have found that wild-type NQO1, but not the inactive polymorphic NQO1, can stabilize endogenous as well as transfected wild-type p53. NQO1-mediated p53 stabilization was especially prominent under induction of oxidative stress. NQO1 also partially inhibited p53 degradation mediated by the human papilloma virus E6 protein, but not when mediated by Mdm-2. Inhibitors of heat shock protein 90 (hsp90), radicicol and geldanamycin, induced degradation of p53 and suppressed p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Differences in the effectiveness of dicoumarol and hsp90 inhibitors to induce p53 degradation and suppress apoptosis in these cell types indicate that NQO1 and hsp90 stabilize p53 through different mechanisms. Our results indicate that NQO1 has a distinct role in the regulation of p53 stability, especially in response to oxidative stress. The present data on the genetic and pharmacologic regulation of the level of p53 have clinical implications for tumor development and therapy.

Lysosomal destabilization in p53-induced apoptosis

The tumor suppressor wild-type p53 can induce apoptosis. M1-t-p53 myeloid leukemic cells have a temperature-sensitive p53 protein that changes its conformation to wild-type p53 after transfer from 37°C to 32°C. We have now found that these cells showed an early lysosomal rupture after transfer to 32°C. Mitochondrial damage, including decreased membrane potential and release of cytochrome c, and the appearance of apoptotic cells occurred later. Lysosomal rupture, mitochondrial damage, and apoptosis were all inhibited by the cytokine IL-6. Some other compounds can also inhibit apoptosis induced by p53. The protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone inhibited the decrease in mitochondrial membrane potential and cytochrome c release, the Ca2+-ATPase inhibitor thapsigargin inhibited only cytochrome c release, and the antioxidant butylated hydroxyanisole inhibited only the decrease in mitochondrial membrane potential. In contrast to IL-6, these other compounds that inhibited some of the later occurring mitochondrial damage did not inhibit the earlier p53-induced lysosomal damage. The results indicate that apoptosis is induced by p53 through a lysosomal-mitochondrial pathway that is initiated by lysosomal destabilization, and that this pathway can be dissected by using different apoptosis inhibitors. These findings on the induction of p53-induced lysosomal destabilization can also help to formulate new therapies for diseases with apoptotic disorders.

Mdm-2 and ubiquitin-independent p53 proteasomal degradation regulated by NQO1

The tumor suppressor p53 is a labile protein whose level is known to be regulated by the Mdm-2–ubiquitin–proteasome degradation pathway. We have found another pathway for p53 proteasomal degradation regulated by NAD(P)H quinone oxidoreductase 1 (NQO1). Inhibition of NQO1 activity by dicoumarol induces p53 and p73 proteasomal degradation. A mutant p53 (p53[22,23]), which is resistant to Mdm-2-mediated degradation, was susceptible to dicoumarol-induced degradation. This finding indicates that the NQO1-regulated proteasomal p53 degradation is Mdm-2-independent. The tumor suppressor p14ARF and the viral oncogenes SV40 LT and adenovirus E1A that are known to stabilize p53 inhibited dicoumarol-induced p53 degradation. Unlike Mdm-2-mediated degradation, the NQO1-regulated p53 degradation pathway was not associated with accumulation of ubiquitin-conjugated p53. In vitro studies indicate that dicoumarol-induced p53 degradation was ubiquitin-independent and ATP-dependent. Inhibition of NQO1 activity in cells with a temperature-sensitive E1 ubiquitin-activating enzyme induced p53 degradation and inhibited apoptosis at the restrictive temperature without ubiquitination. Mdm-2 failed to induce p53 degradation under these conditions. Our results establish a Mdm-2- and ubiquitin-independent mechanism for proteasomal degradation of p53 that is regulated by NQO1. The lack of NQO1 activity that stabilizes a tumor suppressor such as p53 can explain why humans carrying a polymorphic inactive NQO1 are more susceptible to tumor development.

Different locations of carbohydrate-containing sites in the surface membrane of normal and transformed mammalian cells

SummaryA soybean agglutinin was found to agglutinate mouse, rat and human cell lines transformed by viral carcinogens, but not hamster cells transformed by viral or non-viral carcinogens. Normal cells from which the transformed cells were derived were not agglutinated by this agglutinin, but they were rendered agglutinable after short incubation with trypsin or pronase. The transformed hamster cells, on the other hand, became agglutinable only after prolonged treatment with pronase. The agglutination was specifically inhibited by N-acetyl-d-galactosamine, indicating that N-acetyl-d-galactosamine-like saccharides are part of the receptor sites for soybean agglutinin on the surface membrane. Such sites exist in a cryptic form in normal cells; they are exposed in transformed mouse, rat and human cells, but become less accessible in transformed hamster cells. The receptor sites for soybean agglutinin differ from the receptors for two other plant agglutinins (wheat germ agglutinin that interacts with N-acetyl-d-glucosamine-like sites and Concanavalin A that interacts with α-d-glucopyranoside-like sites) which become exposed upon transformation of all lines tested. In normal hamster cells, the receptors for all three agglutinins become exposed after incubation with trypsin, but the exposure of N-acetyl-d-galactosamine-like sites requires the longest enzyme treatment. The results indicate a difference in the location of different carbohydrate-containing sites in the surface membrane. The differences in the exposure of carbohydrate-containing sites in the membrane could not be correlated with the levels of carbohydrate-splitting glycosidases in normal and transformed cells.

Thymic abnormalities and enhanced apoptosis of thymocytes and bone marrow cells in transgenic mice overexpressing Cu/Zn-superoxide dismutase: implications for Down syndrome.

The copper‐zinc superoxide dismutase (CuZnSOD) gene resides on chromosome 21 and is overexpressed in Down syndrome (DS) patients. Transgenic CuZnSOD mice with elevated levels of CuZnSOD were used to determine whether, as in DS, overexpression of CuZnSOD was also associated with thymus and bone marrow abnormalities. Three independently derived transgenic CuZnSOD strains had abnormal thymi showing diminution of the cortex and loss of corticomedullary demarcation, resembling thymic defects in children with DS. Transgenic CuZnSOD mice were also more sensitive than control mice to in vivo injection of lipopolysaccharide (LPS), reflected by an earlier onset and enhanced apoptotic cell death in the thymus. This higher susceptibility to LPS‐induced apoptosis was associated with an increased production of hydrogen peroxide and a higher degree of lipid peroxidation. When cultured under suboptimal concentrations of interleukin 3 or in the presence of tumour necrosis factor, bone marrow cells from transgenic CuZnSOD mice produced 2‐ to 3‐fold less granulocyte and macrophage colonies than control. The results indicate that transgenic CuZnSOD mice have certain thymus and bone marrow abnormalities which are similar to those found in DS patients, and that the defects are presumably due to an increased oxidative damage resulting in enhanced cell death by apoptosis.

Control of lysozyme induction in the differentiation of myeloid leukemic cells

A system has been developed with clones of mouse myeloid leukemic cells in culture to study the induction of synthesis and secretion of lysozyme in relation to other steps in myeloid cell differentiation. Lysozyme was initially absent in all the clones studied. The different clones can be divided into three types according to their ability to be induced to undergo normal cell differentiation by the protein inducer MGI (macrophage and granulocyte inducer). One type of clone that can be induced by MGI to form Fc and C3 receptors and differentiate to mature macrophages and granulocytes (MGI+D+) was also induced by MGI to synthesize and secrete lysozyme. Lysozyme was induced after Fc and C3 receptors, and labeling with 35S-methionine has shown that the induced lysozyme was newly synthesized. MGI+D+ clones were also induced to synthesize and secrete lysozyme by dexamethasone, prednisolone, cytosine arabinoside, or thymidine and in one of four clones by dimethylsulfoxide but not by sodium butyrate. Inhibition of cell multiplication by itself was not sufficient to induce lysozyme synthesis. A second type of clone which can be induced by MGI to form Fc and C3 receptors but not mature cells (MGI+D-) was more weakly inducible by MGI for lysozyme and was not inducible by any of the other compounds. A third type of clone (MGI-D) MGI for receptors or mature cells. One of four MGI-D- clones was induced to synthesize but not secrete lysozyme by dexamethasone together with sodium butyrate, but there was no lysozyme induction by MGI or any of the other compounds separately. The different clones maintained their different properties for at least 6 months in culture. The results indicate that clones with different hereditary blocks in the ability to be induced to differentiate by specific compounds can be used to dissect the process of myeloid cell differentiation, that the sequence of differentiation is induction of Fc and C3 receptors leads to lysozyme leads to mature cells, and that there are separate controls for these developmental steps.

Cytokine control of developmental programs in normal hematopoiesis and leukemia

The establishment of a system for in vitro clonal development of hematopoietic cells made it possible to discover the cytokines that regulate hematopoiesis. These cytokines include colony stimulating factors and others, which interact in a network, and there is a cytokine cascade which couples growth and differentiation. A network allows considerable flexibility and a ready amplification of response to a particular stimulus. A network may also be necessary to stabilize the whole system. Cells called hematopoietic stem cells (HSC) can repopulate all hematopoietic lineages in lethally irradiated hosts, and under appropriate conditions give rise to neuronal, muscle, and epithelial cells. Granulocyte colony stimulating factor induces migration of both HSC and in vitro colony forming cells from the bone marrow to peripheral blood. Granulocyte colony stimulating factor is also used clinically to repair irradiation and chemotherapy associated suppression of normal hematopoiesis in cancer patients, and to stimulate normal granulocyte develpment in patients with infantile congenital agranulocytosis. It is suggested that there may also be appropriate conditions under which in vitro colony forming cells have a wider differentiation potential similar to that shown by HSC. An essential part of the developmental program is cytokine suppression of apoptosis by changing the balance in expression of apoptosis inducing and suppressing genes. Decreasing the level of cytokines that suppress therapeutic induction of apoptosis in malignant cells can improve cancer therapy. Cytokines and some other compounds can reprogram abnormal developmental programs in leukemia, so that the leukemic cells differentiate to mature non dividing cells, and this can also be used for therapy. There is considerable plasticity in the developmental programs of normal and malignant cells.

DNA rearrangement of a homeobox gene in myeloid leukaemic cells.

A homeobox gene rearrangement has been detected in WEHI‐3B mouse myeloid leukaemic cells. The rearranged gene was identified as Hox‐2.4 which is a member of the Hox‐2 gene cluster on mouse chromosome 11. Both the normal and the rearranged genes were cloned and analysed, and the rearranged genomic Hox‐2.4 gene was sequenced. The results indicate that the rearrangement is due to insertion of an intracisternal A particle 5′ upstream to Hox‐2.4 and that this resulted in constitutive expression of the homeobox gene. It is suggested that constitutive expression of the homeobox gene may interrupt the normal development program in these leukaemic cells.

Inhibition of lectin agglutinability by fixation of the cell surface membrane

Abstract Formation of clusters of Concanavalin A binding sites on the surface membrane of lymphoma cells is inhibited by aldehyde fixation of the fluid state of the membrane. Fixation also inhibits cell agglutination by soluble Concanavalin A and binding of cells to Sepharose-conjugated Concanavalin A beads, although three is a similar binding of radioactively labeled concanavalin A molecules to fixed and unfixed cells. Movement of concanavalin A sites on the surface membrane to form clusters is therefore required for cell agglutination. Different degrees of membrane fixation by aldehydes inhibit agglutination by the lectins from wheat germ and soybean.