Apoptosis in megaloblastic anemia occurs during DNA synthesis by a p53-independent, nucleoside-reversible mechanism

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3249-3255 ◽  
Author(s):  
Mark J. Koury ◽  
James O. Price ◽  
Geoffrey G. Hicks
Keyword(s):  
Dna Synthesis ◽  
De Novo ◽  
Megaloblastic Anemia ◽  
Experimental Studies ◽  
Hematopoietic Cells ◽  
S Phase ◽  
De Novo Synthesis ◽  
Complete Reversal ◽  
Phase Accumulation

Abstract Deficiency of folate or vitamin B12 (cobalamin) causes megaloblastic anemia, a disease characterized by pancytopenia due to the excessive apoptosis of hematopoietic progenitor cells. Clinical and experimental studies of megaloblastic anemia have demonstrated an impairment of DNA synthesis and repair in hematopoietic cells that is manifested by an increased percentage of cells in the DNA synthesis phase (S phase) of the cell cycle, compared with normal hematopoietic cells. Both folate and cobalamin are required for normal de novo synthesis of thymidylate and purines. However, previous studies of impaired DNA synthesis and repair in megaloblastic anemia have concerned mainly the decreased intracellular levels of thymidylate and its effects on nucleotide pools and misincorporation of uracil into DNA. An in vitro model of folate-deficient erythropoiesis was used to study the relationship between the S-phase accumulation and apoptosis in megaloblastic anemia. The results indicate that folate-deficient erythroblasts accumulate in and undergo apoptosis in the S phase when compared with control erythroblasts. Both the S-phase accumulation and the apoptosis were induced by folate deficiency in erythroblasts fromp53 null mice. The complete reversal of the S-phase accumulation and apoptosis in folate-deficient erythroblasts required the exogenous provision of specific purines or purine nucleosides as well as thymidine. These results indicate that decreased de novo synthesis of purines plays as important a role as decreased de novo synthesis of thymidylate in the pathogenesis of megaloblastic anemia.

Apoptosis in megaloblastic anemia occurs during DNA synthesis by a p53-independent, nucleoside-reversible mechanism

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3249-3255
Author(s):  
Mark J. Koury ◽  
James O. Price ◽  
Geoffrey G. Hicks
Keyword(s):  
Dna Synthesis ◽  
De Novo ◽  
Megaloblastic Anemia ◽  
Experimental Studies ◽  
Hematopoietic Cells ◽  
S Phase ◽  
De Novo Synthesis ◽  
Complete Reversal ◽  
Phase Accumulation

Deficiency of folate or vitamin B12 (cobalamin) causes megaloblastic anemia, a disease characterized by pancytopenia due to the excessive apoptosis of hematopoietic progenitor cells. Clinical and experimental studies of megaloblastic anemia have demonstrated an impairment of DNA synthesis and repair in hematopoietic cells that is manifested by an increased percentage of cells in the DNA synthesis phase (S phase) of the cell cycle, compared with normal hematopoietic cells. Both folate and cobalamin are required for normal de novo synthesis of thymidylate and purines. However, previous studies of impaired DNA synthesis and repair in megaloblastic anemia have concerned mainly the decreased intracellular levels of thymidylate and its effects on nucleotide pools and misincorporation of uracil into DNA. An in vitro model of folate-deficient erythropoiesis was used to study the relationship between the S-phase accumulation and apoptosis in megaloblastic anemia. The results indicate that folate-deficient erythroblasts accumulate in and undergo apoptosis in the S phase when compared with control erythroblasts. Both the S-phase accumulation and the apoptosis were induced by folate deficiency in erythroblasts fromp53 null mice. The complete reversal of the S-phase accumulation and apoptosis in folate-deficient erythroblasts required the exogenous provision of specific purines or purine nucleosides as well as thymidine. These results indicate that decreased de novo synthesis of purines plays as important a role as decreased de novo synthesis of thymidylate in the pathogenesis of megaloblastic anemia.

scholarly journals Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a.

1991 ◽  
Vol 115 (5) ◽  
pp. 1275-1282 ◽  
Author(s):  
T Usui ◽  
M Yoshida ◽  
K Abe ◽  
H Osada ◽  
K Isono ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
De Novo ◽  
S Phase ◽  
Continuous Treatment ◽  
Protein Kinase Activity

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.

Bioactive Alkaloids from the Tropical Marine Sponge Axinella carteri

1995 ◽  
Vol 50 (9-10) ◽  
pp. 669-674 ◽  
Author(s):  
A. Supriyono ◽  
B. Schwarz ◽  
V. Wray ◽  
L. Witte ◽  
W. E. G. Müller ◽  
...  
Keyword(s):  
Marine Sponge ◽  
Insecticidal Activity ◽  
De Novo ◽  
The Philippines ◽  
De Novo Synthesis ◽  
Mouse Lymphoma Cells ◽  
Guanidine Alkaloids ◽  
Neonate Larvae ◽  
Mouse Lymphoma

Abstract Analysis of the tropical marine sponge Axinella carteri afforded six unusual alkaloids, including the new brominated guanidine derivative 3-bromo-hymenialdisine. The structure elucidation of the new alkaloid is described. The alkaloid patterns of sponges collected in Indonesia or in the Philippines were shown to be qualitatively identical suggesting de novo synthesis by the sponge or by endosymbiontic microorganisms rather than uptake by filterfeeding. All alkaloids were screened for insecticidal activity as well as for cytotoxicity. The guanidine alkaloids hymenialdisine and debromohymenialdisine exhibited insecticidal activity towards neonate larvae of the polyphagous pest insect Spodoptera littoralis (LD50s of 88 and 125 ppm, respectively), when incorporated into artificial diet and offered to the larvae in a chronic feeding bioassay. The remaining alkaloids, including the new compound, were inactive in this bioassay. Cytotoxicity was studied in vitro using L5178y mouse lymphoma cells. Debromohymenialdisine was again the most active compound (ED50 1.8 μg/ml) followed by hymenialdisine and 3-bromohymenialdisine, which were essentially equitoxic and exhibited ED50s of 3.9 μg/ml in both cases. The remaining alkaloids were inactive against this cell line

Immunology: Evidence for the in-vitro de-novo synthesis of immunoglobulin and a previously undescribed 17 kDa protein (TEP-2) by the mucosa of the Fallopian tube

1993 ◽  
Vol 8 (8) ◽  
pp. 1199-1202 ◽  
Author(s):  
S.D. Maguiness ◽  
K. Shrimanker ◽  
O. Djahanbakhch ◽  
B. Teisner ◽  
J.G. Grudzinskas
Keyword(s):  
Fallopian Tube ◽  
De Novo ◽  
De Novo Synthesis

Stretch-mediated visceral smooth muscle growth in vitro

1992 ◽  
Vol 262 (5) ◽  
pp. R895-R900
Author(s):  
O. M. Karim ◽  
K. Pienta ◽  
N. Seki ◽  
J. L. Mostwin
Keyword(s):  
Smooth Muscle ◽  
Dna Synthesis ◽  
De Novo ◽  
Specific Activity ◽  
Muscle Growth ◽  
Mechanical Stimulus ◽  
Trophic Factors ◽  
Taenia Coli ◽  
Visceral Smooth Muscle

An in vitro model of smooth muscle stretch was developed to study mechanical stimulus as a possible mediator of visceral smooth muscle growth and differences in the growth response of smooth muscle from young and old animals. De novo DNA synthesis as measured by the aphidicolin-sensitive specific activity of DNA was used as an index of cell growth. Compared with old tissue, the rate of aphidicolin-sensitive DNA synthesis in smooth muscle from young animals was 3-5 and 1.5-2 times greater in bladder and taenia coli, respectively. Stretch of bladder muscle and taenia coli strips from young animals for 6 h increased the aphidicolin-sensitive specific activity of DNA 3-fold (P less than 0.01) and 1.5-fold (P less than 0.01), respectively. Tissue from old animals, however, under the same conditions increased the rate of aphidicolin-resistant DNA synthesis, possibly implying DNA repair. Autoradiography showed only labeled myocyte nuclei. These results indicate that homeostatic mechanisms modulating myocyte growth in visceral smooth muscle can respond to mechanical stimulus in the absence of other trophic factors.

scholarly journals Release of interleukin-1 and interleukin-6 from human monocytes by antithymocyte globulin: requirement for de novo synthesis

Blood ◽  
1992 ◽  
Vol 80 (10) ◽  
pp. 2531-2538 ◽  
Author(s):  
P Rameshwar ◽  
P Gascon
Keyword(s):  
Antithymocyte Globulin ◽  
De Novo ◽  
Interleukin 1 ◽  
Immunosuppressive Agent ◽  
Biologically Active ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
De Novo Synthesis ◽  
Hematopoietic Growth Factors

Abstract Antithymocyte globulin (ATG) is an effective treatment in patients with severe aplastic anemia (SAA). Its mechanism of action remains unclear, although it has been assumed to be immunosuppressive. However, ATG has also been shown by several laboratories to be immunostimulatory. Recently, interleukin-1 (IL-1) production has been found to be decreased in lipopolysaccharide-stimulated peripheral blood monocytes obtained from SAA patients. We have investigated the ability of ATG to function as an immunostimulatory agent via the production of IL-1 and IL-6 by normal human monocytes in vitro. Supernatants from ATG- stimulated monocytes were assayed for biologically active and immunoreactive IL-1 and IL-6. We have found that ATG, via its F(ab')2 fragment is a powerful inducer of IL-1 and IL-6 production. Furthermore, ATG induction of both cytokines from normal monocytes required de novo synthesis, as determined by 35S-methionine incorporation. Because these two cytokines synergize with other cytokines at both the stem cell and progenitor levels, these stimulatory properties of ATG may be relevant to the treatment of SAA. This would favor the hypothesis of a bimodal mechanism for ATG as an inducer of hematopoietic growth factors and as an immunosuppressive agent.

scholarly journals Congenital Familial Megaloblastic Anemia

Blood ◽  
1971 ◽  
Vol 37 (6) ◽  
pp. 615-623 ◽  
Author(s):  
BEATRICE C. LAMPKIN ◽  
ALLAN PYESMANY ◽  
CAROL B. HYMAN ◽  
DENMAN HAMMOND
Keyword(s):  
Bone Marrow ◽  
Folic Acid ◽  
Dna Synthesis ◽  
Vitamin B12 ◽  
Megaloblastic Anemia ◽  
Vitamin Deficiency ◽  
Preliminary Step ◽  
Orotic Aciduria

Abstract Two sisters with a previously unreported megaloblastic anemia unassociated with a deficiency of either folic acid or vitamin B12 are described. Deficiencies of these vitamins were ruled out by standard studies. All other previously reported forms of megaloblastic anemia not secondary to a vitamin deficiency, such as orotic aciduria, were also excluded by appropriate studies. Optimal hemoglobin responses were obtained after the administration of large amounts of both vitamin B12 and folic acid. Because of this hemoglobin response, the conversion of deoxyuridine-5-monophosphate to deoxythymidine-5-monophosphate in vitro was examined in bone marrow samples from both patients using a modification of a method described by Killmann.18 This preliminary step in DNA synthesis was found to be normal. The results of this test and the optimal hemoglobin response after administration of both vitamins suggest that both folic acid and vitamin B12 may be necessary at some other preliminary step in DNA synthesis.

PDGF and IL-1 induce and activate specific protein kinase C isoforms in mesangial cells

1996 ◽  
Vol 271 (1) ◽  
pp. F108-F113 ◽  
Author(s):  
M. B. Ganz ◽  
B. Saksa ◽  
R. Saxena ◽  
K. Hawkins ◽  
J. R. Sedor
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
De Novo ◽  
Mesangial Cells ◽  
De Novo Synthesis ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Alpha Beta ◽  
Pkc Alpha

In vitro and in vivo data suggest a remarkable plasticity in the differentiated phenotype of intrinsic glomerular cells, which after injury express new structures and functions. We have shown that a protein kinase C (PKC) isoform, beta II, is expressed in diseased but not normal glomeruli. Since intrarenal cytokine synthesis has been implicated in the pathogenesis of progressive glomerular injury, we have hypothesized that these mediators induce a change in isoform profile. To test this hypothesis in vitro, we have determined whether platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) alter the expression or activation of PKC isoforms in cultured mesangial cells (MCs). By immunoblot and ribonuclease (RNase) protection assays, both PDGF and IL-1 induce as early as 2 h de novo synthesis of PKC-beta II. Since MCs constitutively express PKC-alpha, -beta I, and -zeta, we also determined whether IL-1 or PDGF alter the activity of these isoforms. PDGF maximally induced translocation of PKC-alpha (10 min), -beta I (90 min), -epsilon (120 min), and -zeta (120 min) from the cytosolic to the membrane fraction. IL-1, in contrast, did not alter the distribution of alpha, beta I, or epsilon at any time measured but did induce PKC-zeta translocation. These data suggest inflammatory mediators regulate PKC isoform activity in diseased glomeruli both by de novo synthesis of unexpressed isoforms and by activation of constitutively expressed PKC isoforms.

Alterations in the cell cycle of mouse cumulus granulosa cells during expansion and mucification in vivo and in vitro

1996 ◽  
Vol 8 (6) ◽  
pp. 935 ◽  
Author(s):  
AW Schuetz ◽  
DG Whittingham ◽  
R Snowden
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Granulosa Cells ◽  
Dna Content ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Ovarian Follicles ◽  
S Phase

The cell cycle characteristics of mouse cumulus granulosa cells were determined before, during and following their expansion and mucification in vivo and in vitro. Cumulus-oocyte complexes (COC) were recovered from ovarian follicles or oviducts of prepubertal mice previously injected with pregnant mare serum gonadotrophin (PMSG) or a mixture of PMSG and human chorionic gonadotrophin (PMSG+hCG) to synchronize follicle differentiation and ovulation. Cell cycle parameters were determined by monitoring DNA content of cumulus cell nuclei, collected under rigorously controlled conditions, by flow cytometry. The proportion of cumulus cells in three cell cycle-related populations (G0/G1; S; G2/M) was calculated before and after exposure to various experimental conditions in vivo or in vitro. About 30% of cumulus cells recovered from undifferentiated (compact) COC isolated 43-45 h after PMSG injections were in S phase and 63% were in G0/G1 (2C DNA content). Less than 10% of the cells were in the G2/M population. Cell cycle profiles of cumulus cells recovered from mucified COC (oviducal) after PMSG+hCG-induced ovulation varied markedly from those collected before hCG injection and were characterized by the relative absence of S-phase cells and an increased proportion of cells in G0/G1. Cell cycle profiles of cumulus cells collected from mucified COC recovered from mouse ovarian follicles before ovulation (9-10 h after hCG) were also characterized by loss of S-phase cells and an increased G0/G1 population. Results suggest that changes in cell cycle parameters in vivo are primarily mediated in response to physiological changes that occur in the intrafollicular environment initiated by the ovulatory stimulus. A similar lack of S-phase cells was observed in mucified cumulus cells collected 24 h after exposure in vitro of compact COC to dibutyryl cyclic adenosine monophosphate (DBcAMP), follicle-stimulating hormone or epidermal growth factor (EGF). Additionally, the proportion of cumulus cells in G2/M was enhanced in COC exposed to DBcAMP, suggesting that cell division was inhibited under these conditions. Thus, both the G1-->S-phase and G2-->M-phase transitions in the cell cycle appear to be amenable to physiological regulation. Time course studies revealed dose-dependent changes in morphology occurred within 6 h of exposure in vitro of COC to EGF or DBcAMP. Results suggest that the disappearance of the S-phase population is a consequence of a decline in the number of cells beginning DNA synthesis and exit of cells from the S phase following completion of DNA synthesis. Furthermore, loss of proliferative activity in cumulus cells appears to be closely associated with COC expansion and mucification, whether induced under physiological conditions in vivo or in response to a range of hormonal stimuli in vitro. The observations indicate that several signal-transducing pathways mediate changes in cell cycle parameters during cumulus cell differentiation.

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