Studies on the mechanism of tumor inhibition by L-asparaginase. Effects of the enzyme on asparagine levels in the blood, normal tissues, and 6C3HED lymphomas of mice: differences in asparagine formation and utilization in asparaginase-sensitive and -resistant lymphoma cells.

L-asparaginases of agouti serum and Escherichia coli cause a profound lowering in the level of free asparagine in the blood of treated mice and also in the tissues. During treatment, normal tissues and resistant 6C3HED lymphomas survive unharmed with intracellular asparagine levels which are critically low for sensitive lymphomas. An explanation for this contrast between the two types of lymphoma is provided by the finding that resistant cells have not only a higher asparagine synthetic capacity than sensitive cells but appear able to utilize endogenous asparagine preferentially for protein synthesis. Cell-free extracts of resistant cells contain an asparaginase synthetase, but this is not found in preparations from sensitive cells.

Promotion of replication in lymphoid cells by specific thiols and disulfides in vitro. Effects on mouse lymphoma cells in comparison with splenic lymphocytes.

Numerous lines of mouse lymphoid tumors (13 of 22 tested) showed, with increased sensitivity, a property of normal mouse splenic lymphocytes, the potential for growth promotion in vitro by specific thiols added to standard culture media. For lymphoma L1210 (V), structure activity relationships were examined; 9 of 30 thiols promoted growth; the most active was alpha-thioglycerol, effective at 0.2 microM. Thiols became oxidized under conditions of tissue culture and had half-lives of less than 8 h. Disulfides of active thiols promoted growth of lymphoma cells. The mitogenic response of splenic lymphocytes to lectins was increased by thiols-disulfides which promoted the growth of lymphoma cells, but the response varied with the mitogen preparation used and under some conditions thiols-disulfides were inhibitory.

L-asparaginase: inhibition of early mitosis in regenerating rat liver.

L-Asparaginase in agouti serum and in extracts from Escherichia coli inhibits the early wave of mitosis occurring in rat liver approximately 30 hours after hepatectomy, but even with continued treatment of the animal the later wave at 50 hours is not inhibited. This result differs from the permanent inhibition of growth which asparaginase causes in various tumors.

Epigenetic changes in the repression and induction of asparagine synthetase in human leukemic cell lines.

In common with certain other lymphoid neoplasms, cells of the human lymphocytic leukemia lines 1873 and 1929 are asparagine (ASN) auxotrophs. Asparagine synthetase (ASY), which is a housekeeping gene, is repressed and the promoting region of the gene is highly methylated. We now demonstrate in these cells multiple levels in control of the expression of this gene, in a system of cocultivation with macrophages and other cell types. In this system, mediated by cell-to-cell contact, ASY becomes expressed by the leukemic cells and they become prototrophic. Demethylation of ASY occurs; it follows expression and is permanent over multiple cell generations, but the cells return to auxotrophy with rapid repression of ASY on removal from cell contact. With ASY expression, the associated histone H3 at lysine position 9 (H3K9) becomes acetylated and H3K4, methylated. In contrast to other systems, H3K9 methylation does not characterize the repressed state. The changes leading from repression to induction of ASY and demethylation parallel the physiological changes specific to functional maturation of normal lymphoid precursors. The lability of expression of ASY has potential significance in determining the sensitivity of leukemic cells to L-asparaginase.

Arsenic targets tubulins to induce apoptosis in myeloid leukemia cells.

Arsenic exhibits a differential toxicity to cancer cells. At a high concentration (>5 microM), As2O3 causes acute necrosis in various cell lines. At a lower concentration (0.5-5 microm), it induces myeloid cell maturation and an arrest in metaphase, leading to apoptosis. As2O3-treated cells have features found with both tubulin-assembling enhancers (Taxol) and inhibitors (colchicine). Prior treatment of monomeric tubulin with As2O3 markedly inhibits GTP-induced polymerization and microtubule formation in vitro but does not destabilize GTP-induced tubulin polymers. Cross-inhibition experiments indicate that As2O3 is a noncompetitive inhibitor of GTP binding to tubulin. These observations correlate with the three-dimensional structure of beta-tubulin and suggest that the cross-linking of two vicinal cysteine residues (Cys-12 and Cys-213) by trivalent arsenic inactivates the GTP binding site. Furthermore, exogenous GTP can prevent As2O3-induced mitotic arrest.

Methylation of the asparagine synthetase promoter in human leukemic cell lines is associated with a specific methyl binding protein.

We have examined the methylation profiles of the asparagine synthetase (ASY) promoter in a number of human leukemic cell lines in relation to their asparagine (ASN) requirements in vitro. Cells in which the promoter is highly methylated are auxotrophs and express ASY at very low levels. Electromobility shift assays (EMSA) of nuclear extracts with oligomers from the promoting region show, in addition to recognized transcription factor binding, a novel methyl binding protein specific for a 12 base consensus sequence, which includes a single methylated CpG. This sequence overlaps that of the amino-acid response unit of the ASY promoter, which is activated byATF4 and C/EBP. Competition by the methyl binding protein could account for the observed failure of the methylated promoter to bind these transcription factors and consequently, although other mechanisms can also be operative, for the specific repression of the gene. The ASY methyl binding protein (ASMB) is present in leukemic lymphoid and myeloid cells irrespective of their methylation status, and in normal lymphocytes after phytohemagglutinin stimulation. It has been purified by affinity chromatography and has a molecular size of 40 kDa in 10% SDS-polyacrylamide gels.

Molecular characterization of a variant Ph1 translocation t(9;22;11) (q34;q11;q13) in chronic myelogenous leukemia (CML) reveals the translocation of the 3'-part of BCR gene to the chromosome band 11q13.

We performed cloning and sequence analysis of translocation junctions at 11q- and 22q- (Ph1) chromosomes and the corresponding germline DNAs of a variant Ph1-positive CML with t(9;22;11)(q34;q11;q13). Southern blot analysis using probes for different regions of bcr mapped the translocation break near the 5'-side of bcr exon 4. Cloning, Southern blot analysis and restriction map analysis of both bcr fragments showed that the part of bcr 3'- to the translocation break moved to 11q13. Sequence analysis of the translocation junction on the Ph1 chromosome showed that the translocation break occurred 63 bp upstream of exon 4. Compared to the germline sequence, bcr sequence from the translocated partners showed deletion of seven basepairs at the site of translocation. A probe derived from the 5'-region of the clone isolated from the 11q- chromosome identified clonal rearrangements in the leukemic DNA. Restriction map and sequence analysis showed that this clone consisted of the 3'-half of the glutathione S-transferase Pi (GST-Pi) gene and the 3'-part of bcr. We identified two point mutations in the GST-Pi allele involved in translocation. Northern blot analysis showed that the GST-Pi gene was expressed in the leukemic cells at blast crisis but not at chronic phase; however, no fusion mRNA between GST-Pi and bcr was identified. We did not find any sequence homology between 11q13 DNA and 22q11 DNA around the translocation breakpoints; however, sequences homologous to ALU repeats were identified close to the sites of translocation breaks at 22q11 and 11q13. This study supports our hypothesis that variant Ph1 translocations may occur as primary cytogenetic changes similar to the classical Ph1 translocations.

Clinical and morphological features of cases of trisomy 13 in acute non-lymphocytic leukemia.

Trisomy 13 has been infrequently reported as a primary non-random karyotypic change in myeloid leukemias. To elucidate its clinical significance we examined the clinical and hematological data in nine ANLL patients in whom we found this change, in a series of 175 cytogenetically abnormal ANLL patients. Morphologically, six of the patients were FAB-M1, two were FAB-M4 and one was FAB-M5. Bone marrow aspirates contained more than 90% blasts in eight of the patients. By immunophenotype, TdT was present in four of the patients, CD34 was present in four of five patients tested and CD5 was present in one of five patients tested. Blast cells in all patients expressed two or more myeloid surface antigens. These data suggest the proliferation of an immature myeloid cell in these patients. Complete remission was achieved in seven patients; however, remissions were short-lived. Eight patients expired between 1 and 13 months from diagnosis (median survival 5 months). Combining our findings with data in the published literature on trisomy 13 in ANLL, a larger data set consisting of 29 patients was established to determine better the clinical significance of this cytogenetic entity in ANLL. We found that this cytogenetic change has been reported in all subsets of FAB classification excepting M6 and M7. Median age at presentation was 60 years and no association with gender was noted. Median WBC was 29.5 x 10(9)/l, the majority of patients were thrombocytopenic (median platelet count 86 x 10(9)/l) and median survival was 5.2 months. This study associates trisomy 13 with malignant transformation of myeloid progenitor cells. These patients respond well to induction therapy, but relapse occurs quickly and the survival duration is poor.

Fluoroscopically guided hysteroscopic division of adhesions in severe Asherman syndrome.

BACKGROUND: Severe Asherman syndrome that is stage III disease according to the American Fertility Society, with obliteration of the uterine cavity and the inability to visualize isolated pockets of the intrauterine cavity, makes safe and effective hysteroscopic division of adhesions difficult, if not impossible. TECHNIQUE: A 16-gauge, 80-mm Tuohy needle is introduced into the endocervical canal alongside a 5-mm diagnostic hysteroscope. The surgeon probes the area beyond the adhesion with the needle. Ultravist 76.9% is injected through the needle under fluoroscopic and hysteroscopic control. Hidden pockets of endometrium can be located radiographically, a passageway is created using the needle, and subsequent division of adhesions is performed under direct vision with hysteroscopic scissors. EXPERIENCE: Since 1984, approximately 55 women with severe Asherman syndrome have undergone this procedure. All patients required at least two procedures, and one woman required six. There have been two cases of uneventful perforation with the Tuohy needle, and all women resumed menstruation. No serious complications have occurred. CONCLUSION: This technique provides an intraoperative fluoroscopic view of pockets of endometrium behind an otherwise blind-ending endocervical canal in women with severe Asherman syndrome, allowing guided division of adhesions and reducing the likelihood of perforation and formation of false passageways.

Glial cell line-derived neurotrophic factor protects midbrain dopamine neurons from the lethal action of the weaver gene: a quantitative immunocytochemical study.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect and repair midbrain dopamine neurons in vivo using animal models created with neurotoxins. The weaver mouse (wv/wv) has natural and spontaneous midbrain dopaminergic cell death which gives a unique opportunity to examine the effects of GDNF. The present study was designed to investigate a possible neuroprotective role by GDNF for midbrain dopamine neurons in the wv/wv. Weaver pups were given 1 microl injections on postnatal day 1. The wv/wv placebo group received a single unilateral injection into the right lateral ventricle of phosphate buffered saline (PBS) while the GDNF treated wv/wv mice received either 1.0 microg/microl or 10.0 microg/microl GDNF in PBS. All mice were sacrificed on postnatal day 20 and their brains were processed for tyrosine hydoxylase (TH) immunocytochemistry. When compared to the placebo group, the 1 microg GDNF group showed significantly less cell death on the injection side, but the contralateral side showed no significant sparing of TH neurons. The combined counts from both sides show significantly more TH staining neurons in the 1 microg GDNF group compared to placebo. When compared to placebo-injected controls, the 10 microg GDNF treated group showed significantly more TH staining neurons on the injected side, contralateral side, and combined. The results demonstrate that GDNF does protect weaver dopaminergic midbrain neurons from the lethal action of the weaver gene and the effect is positively correlated to dosage.

Studies of the mechanism of growth promotion of lymphoma cells by 2-mercaptoethanol in vitro.

Rates of incorporation of thymidine and uridine, but not leucine, decrease markedly in L1210 (V) cells within 1 hour of incubation in DULBECCO'S medium containing 10% serum. 2-Mercaptoethanol (2-ME) after a latent period of more than 5 hours causes an increase in nucleotide incorporation. Bovine serum albumin can substitute for serum in the medium, but a higher concentration of 2-ME is required for growth. After charcoal treatment of the albumin less 2-ME is required. These experiments suggest that inhibition of the tumor cells is caused by a serum factor whose effect is antagonized by the thiol.

L1210(A) mouse lymphoma cells depleted of glutathione with L-buthionine-S-R-sulfoximine proliferate in tissue culture.

L1210(A) mouse lymphoma cells have been adapted to long-term tissue culture in the presence of L-buthionine-S-R-sulfoximine in concentrations of 1-10 mM. As a result of the inhibitory action of this compound on the synthesis of gamma-glutamylcysteine, the dipeptide precursor of glutathione, the cells are depleted of more than 90% of their normal cellular glutathione content. The residual 10% seems to resist depletion at high concentrations of buthionine sulfoximine. Glutathione depleted cells proliferate at a rate similar to that of non-depleted cells, and show full viability. Upon transfer of cells into inhibitor-free medium, they fully regain their original glutathione content. It is concluded that these cells contain at least two pools of glutathione: a large cytoplasmic pool and a smaller, possibly mitochondrial, pool. It is further concluded that the large pool of cytoplasmic glutathione is not obligatory for cell growth and mitosis.

Mercaptoethanol protects glutathione depleted cells.

Buthionine sulfoximine depleted the glutathione (GSH) level of mouse lymphoma L1210A cells in culture to 6% of control and killed the cells within 48 hours in medium supplemented with fetal calf serum or bovine serum albumin. Mercaptoethanol or alpha-thioglycerol but not GSH or cysteine added to the medium protected the cells from the effect of GSH depletion. Horse serum was also protective, and this effect was removed by dialysis over 65 hours and could not be restored by adding GSH. Mercaptoethanol alone had a protective action in the dialyzed sera. The results suggest that mercaptoethanol may act independently and perform the functions of GSH.

Production of autoinhibitory factors by mouse lymphoma cells in vitro and its relationship to thiol dependence.

Five different kinds of mouse lymphoma cells conditioned culture medium so that it became inhibitory to thiol-dependent cells but not to thiol-independent cells. Conditioning was rapid and appeared to be complete in 1-3 hr. The inhibitory effects could be reversed by concentrations of 2-mercaptoethanol higher than those usually required for growth promotion. Inhibitory factors were concentrated by ultrafiltration and chromatography on Sephadex G-10 which showed their apparent molecular weights to be 300-600. The factors were stable at 100 degrees and to proteases but were inactivated (after reduction) by iodoacetamide, indicating that they may be thiols or disulfides.

Macromolecular inhibitory factor for lymphoid cells produced by mouse macrophages.

Supernatants of high density cultures of mouse peritoneal macrophages were inhibitory to homologous lymphoid cells in vitro by a number of parameters: proliferation and survival of lymphoma cells was impaired, the immune response of spleen cells to sheep red cells and to Concanavalin A (Con A) was decreased. Inhibition was due to factor(s) with mol. wt of approximately 110,000 as estimated by gel filtration. Activity was stable to heating at 56 degrees, and was resistant to trypsin but not to pronase.