Poly(A) polymerase (PAP; EC 2.7.7.19) catalyzes mRNA polyadenylation. Its activity and isoform levels vary during cell cycle transformation and apoptosis. It has become widely accepted that cell death after DNA damage by anticancer agents is primarily the result of apoptosis and that cells able to evade apoptosis will be resistant to cell killing. The therapeutic agents interferon (IFN), 5-fluorouracil (5-FU) and tamoxifen (Tam) with different mechanisms of action mediate both partial dephosphorylation and inactivation of PAP, detected by immunoblotting analysis and PAP enzyme assay, respectively. We examined the apoptotic tendencies of HeLa and WISH cell lines caused by one of the drugs used, 5-FU. The trend in the cells examined, observed by DAPI and/or DNA fragmentation assay, was found to be accompanied by and reversibly related to PAP activity levels and PAP lower mobility phosphorylated forms of 106 and 100 kDa isoforms. Moreover, a cell type-modulated, differential response of HeLa (chemosensitive cells) versus WISH (drug-resistant diploid cells) has been revealed. This finding yields information on the possible use of PAP as a tumor marker involved in cell commitment and/or induction of apoptosis and may help to improve our understanding of tumor cell sensitivity to anticancer agents.
PatelT., GoresG.J., KaufmannS.H.The role of proteases during apoptosis.FASEB J1996; 10: 587–97.
8.
WangJ.Y.J.Oncoprotein phosphorylation and cell cycle.Biochim Biophys Acta1992; 1114: 179–92.
9.
WahleE.3'-end cleavage and polyadenylation of mRNA precursors.Biochim Biophys Acta1995; 1261, 183–94.
10.
JacksonR.J.Cytoplasmic regulation of mRNA function: the importance of the 3’ untranslated region.Cell1993; 74: 9–14.
11.
JacobsonA., PeltzS.W.Interrelationships of the pathways of mRNA decay and translation in eukaryotic cell.Annu Rev Biochem1996; 65: 693–739.
12.
CarpousisA.J., VenzoN.F., RayalL.C.mRNA degradation: a tale of poly(A) and multiprotein machines.Trends Gen1999; 15: 24–8.
13.
ZhaoW., ManleyJ.L.Complex alternative RNA processing generated an unexpected diversity of poly(A) polymerase isoforms.Mol Cell Biol1996; 16: 2378–86.
14.
RaabeT., BollumF.J., ManleyJ.L.Primary structure and expression of bovine poly(A) polymerase.Nature1991; 353: 229–34.
15.
MartinG., KellerW.Mutational analysis of mammalian poly(A) polymerase identifies a region for primer binding and a catalytic domain, homologous to the family X polymerases and other nucleotidyltranferases.EMBO J1996; 15: 2593–603.
16.
ColganD.F., MurthyK.G.K., PrivesC., ManleyJ.L.Cell-cycle regulated regulation of poly(A) polymerase by phosphorylation.Nature1996; 384: 282–4.
17.
ColganD.F., MurthyK.G.K., ZhaoW., PrivesC., ManleyJ.L.Inhibition of poly(A) polymerase requires p34cdc2/cyclin B phosphorylation of multiple consensus and non-consensus sites.EMBO J1998; 17: 1053–62.
18.
KankiJ.P., NewportJ.W.The cell cycle dependence of protein synthesis during Xenopus laevis development.Dev Biol1991; 146: 198–213.
19.
GundersonS.I., VagnerS., Polycarpou-SchwarzM., MattajI.W.Involvement of the carboxyl terminus of vertebrate poly(A) polymerase in U1A autoregulation and in the coupling of splicing and polyadenylation.Genes Dev1997; 11: 761–73.
20.
AtabasidesH., TsiapalisC.M., HavredakiM.Dephosphorylation, proteolysis and reduced activity of poly(A) polymerase associated with U937 cell apoptosis.Exp Cell Res1998; 244: 433–40.
21.
BergerS.L., CooperH.L.Very short-lived and stable mRNA from resting human lymphocytes.Proc Natl Acad Sci USA1975; 72: 3873–7.
22.
CarmichaelJ., DeGraffW.G., GazdarA.F., MinnaJ.D., MitchellJ.B.Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing.Cancer Res1987; 47: 936–42.
23.
LowryO.H., RosebroughN.L., FarrA.L., RandallR.J.Protein measurement with the Folin phenol reagent.J Biol Chem1951; 193: 265–75.
24.
LaemmliU.K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature1970; 227: 680–5.
25.
BurnetteN.W.Western blotting. Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.Anal Biochem1981; 112: 195–203.
26.
BlakeM.S., JohnstonK.H., Russel-JonesG.J., GothschlichE.G.A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on western blots.Anal Biochem1984; 136: 175–9.
27.
ThuressonA.C., AströmJ., AströmA., GrönvikK.O., VirtanenA.Multiple forms of poly(A) polymerase in human cells.Proc Natl Acad Sci USA1994; 91: 974–83.
28.
TsiapalisC.M., DorsonJ.W., BollumF.J.Purification of terminal riboadenylate transferase from calf thymus gland.J Biol Chem1975; 250: 4486–96.
29.
TrangasT., CourtisN., PangalisG.A.Polyadenylic acid polymerase in normal and leukemic human leukocytes.Cancer Res1984; 44: 3691–7.
30.
LazebnikY.A., ColeS., CookeC.A., NelsonW.G., EarnshawW.C.Nuclear events in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis.J Cell Biol1993; 123: 7–22.
31.
EastmanA.Assays for DNA fragmentation, endonucleases, and intracellular pH and Ca2+ associated with apoptosis.Methods Cell Biol1995; 46: 41–55.
32.
TsiapalisC.M.Regulation of mRNA polyadenylation-deadenylation.Mol Biol Rep1987; 12: 291–8.
33.
BeelmanC.A., ParkerR.Degradation of mRNA in eukaryotes.Cell1995; 81: 179–83.
34.
SachsA.B., SarnowP., HentzeM.W.Starting at the beginning, middle and end: Translation initiation in eukaryotes.Cell1997; 89: 831–8.
35.
RajagopalanL., MalterL.S.Regulation of eukaryotic messenger RNA turnover.Prog Nucl Acid Res Mol Biol1997; 56: 257–85.
36.
WahleE., KellerW.The biochemistry of polyadenylation.Trends Biol Sci1996; 21: 247–50.
37.
EdmondsM.Polyadenylate polymerase. In: Dahlberg JE, Abelson JN, eds. Methods in enzymology. RNA processing, part B.Academic Press, Inc., 1990; 181: 161–70.
38.
KyriakopoulouC., TsiapalisC.M., HavredakiM.Biochemical and immunological identification and enrichment of poly(A) polymerase from human thymus.Mol Cell Biochem1996; 154: 9–16.
39.
CourtisN., TrangasT., TsiapalisC.M.Increase in the levels of activity of polyadenylic acid metabolizing enzymes following phytohaemagglutinin stimulation of human lymphocytes.Mol Cell Biochem1987; 75: 33–42.
40.
KazazoglouT., TsiapalisC.M., HavredakiM.Polyadenylate polymerase activity in stationary and growing cell cultures.Exp Cell Biol1987; 55: 164–72.
41.
PangalisG.A., TrangasT., RoussouP.A., TsiapalisC.M.The value of poly(A) polymerase as a prognostic biochemical marker in chronic lymphocytic leukemia.Acta Haematol1985; 74: 31–4.
42.
GounarisA., CourtisN., PangalisG.A., TsiapalisC.M., TrangasT.Relation between the activity of poly(A) polymerase and the levels of protein synthesis in the lymphocytes of patients with chronic lymphocytic leukemia.Blut1988; 57: 293–6.
43.
TrangasT., CourtisN., GounarisA.Patterns of adenosine deaminase, ecto-5'-nucleosidase, poly(A) polymerase and surface light chain expression in chronic lymphocytic leukemias.Blut1989; 58: 187–93.
44.
HavredakiM., GaryfallidesS., TsiapalisC.M.Interferon-caused alterations of poly(A) polymerase activity levels in established cell lines.J Biol Regul Homeost Agents1992; 6: 31–2.
45.
HavredakiM., GiannoulakiH., TsiapalisC.M.Poly(A) polymerase from HeLa cells is increased by the antitumor drug bleomycin.Clin Chem Enzym Comms1993; 5: 143–9.
46.
ScorilasA., CourtisN., YotisJ., TallieriM., MichailakisM., TrangasT.Poly(A) polymerase activity levels in breast tumor cytosols.J Exp Clin Cancer Res1998; 17: 511–8.
47.
PickardM., DiveC., KinsellaA.R.Differences in resistance to 5-fluorouracil as a function of cell cycle delay and not apoptosis.Br J Cancer1995; 72: 1389–96.
48.
HuschtshaL.I., BartierW.A., AnderssonR., TattersallM.H.N.Characteristics of cancer cell death after exposure to cytotoxic drugs in vitro.Br J Cancer1996; 73: 54–60.
49.
PritchardD.M., WatsonA.J.M., PottenC.S., JackmanA.L., HickmanJ.A.Inhibition by uridine but not thymine of p53-dependent intestinal apoptosis initiated by 5-fluorouracil: evidence for the involvement of RNA perturbation.Proc Natl Acad Sci USA1997; 94: 1795–9.
50.
GremJ.L.Fluorinated pyrimidines. In: ChabnezB.A., CollingsJ.M., eds. Cancer chemotherapy: Principles and practice.Philadelphia: J.B. Lippincott Co, 1990; 180–226.
51.
GeoffroyF.J., AllegraC.J., SinhaB., GremJ.L.Enhanced cytotoxicity with Interleukin1-alpha and 5-fluorouracil in HCT116 colon cancer cells.Oncol Res1994; 6: 581–91.
52.
LongoG.S., IzzoJ., ChangY.M.Pretreatment of colon carcinoma cells with Tomudex enhances 5-fluorouracil cytotoxicity.Clin Cancer Res1998; 4: 469–73.
53.
DouQ.P., AnB., WillP.L.Induction of a retinoblastoma phosphatase activity by anticancer drugs accompanies p53-independent G1 arrest and apoptosis.Proc Natl Acad Sci USA1995; 92: 9019–23.
54.
DouQ.P., LuiV.W.Y.Failure to dephosphorylate retinoblastoma protein in drug-resistant cells.Cancer Res1995; 55: 5222–5.
55.
TakedaH., HaisaM., NaomotoY.Effect of 5-fluorouracil on cell cycle regulatory proteins in human colon cancer cell line.Jpn J Cancer Res1999; 90: 677–84.
56.
AtabasidesH., TsiapalisC.M., HavredakiM.Poly(A) polymerase specifically implicated in the mechanism of chemotherapeutic drug action during cell apoptosis.Int J Biol Markers2000; 15: 10–4.
