Several tumor suppressor genes have been shown to regulate cellular susceptibility to proliferation or apoptotic cell death. An essential first step in studies with the long-range goal of determining the effect of a tumor suppressor gene on cellular susceptibility to apoptosis is careful characterization of the cell's response to an apoptotic stimulus. The goals of this study were to characterize the apoptotic response of a tuberous sclerosis complex-2 (Tsc2) tumor suppressor gene-null cell line, to establish valid biochemical events that can be used as apoptosis markers, and to determine how these events correlate with apoptosis-specific morphologic changes. For characterization of apoptosis, we treated Tsc2-null renal epithelial tumor cells (ERC-18) with okadaic acid (OKA, 0.1—0.25 μM), and measured the biochemical and morphologic events during the apoptotic response. Electron microscopic and immunocytochemica l evaluation showed an early loss of microvilli and a loss of vinculin and talin staining from focal adhesions within 1 hour. During the first 2 hours of treatment with 0.25 μM OKA, ERC-18 cells rounded and ∼50% detached from the culture vessel with minimal membrane bleb formation. Phosphatidylserine externalization, chromatin margination and fragmentation, cytochrome C release, and caspase-3 and -7 cleavage were evident at 6 hours. Maximal membrane bleb formation occurred between 6 and 10 hours. Cells progressed to secondary oncotic necrosis between 10 and 24 hours of OKA treatment. Almost all cells had an oncotic phenotype after 24 hours, and 17.5% lost cell membrane integrity. A small subpopulation (< 5%) of OKA-treated cells underwent primary oncotic necrosis within 6 hours. Interestingly, the caspase-3 and -7 inhibitor Z-DEVD-FMK did not inhibit or delay OKA-induced apoptosis in these cells. Our results suggest a complex apoptotic model involving 2 or more potentially parallel death pathways. Although caspase-3 and -7 cleavage occurs during apoptosis in this model, this cleavage may not independently regulate cell death in ERC-18 cells. Therefore, measurement of apoptosi s in this model requires analysis of both biochemical and morphologi c events.
Adayev T., Estephan R., Meserole S., Mazza B., Yurkow EJ, BanerjeeP. (1998). Externalization of phosphatidylserine may not be an early signal of apoptosis in neuronal cells, but only the phosphatidylserine-displayin g apoptotic cells are phagocytosed by microglia [published erratum appears in J Neurochem1999 Feb;72(2):886]. J Neurochem71: 1854—1864.
2.
Agarwal ML, Taylor WR, Chernov MV, Chernova OB, Stark GR (1998). The p53 network. J Biol Chem273: 1—4.
3.
Al-Saleem T. , Wessner LL, Scheithauer BW, Patterson K., Roach ES, Dreyer SJ, Fujikawa K., Bjornsson J., Bernstein J., Henske EP (1998). Malignant tumors of the kidney, brain, and soft tissues in children and young adults with the tuberous sclerosis complex. Cancer83: 2208—2216.
4.
Bacso Z., Everson RB, Eliason JF (2000). The DNA of annexin V-binding apoptotic cells is highly fragmented. Cancer Res60: 4623— 4628.
5.
Bernstein J. , Robbins TO (1991). Renal involvement in tuberous sclerosis. Ann NY Acad Sci615: 36—49.
6.
Bonneau MJ, Poulin R. (2000). Spermine oxidation leads to necrosis with plasma membrane phosphatidylserine redistribution in mouse leukemia cells. Exp Cell Res259: 23—34.
7.
Bowen C., Voeller HJ, Kikly K., Gelmann EP (1999). Synthesis of procaspases-3 and -7 during apoptosis in prostate cancer cells. Cell Death Differ6: 394—401.
8.
Brancolini C. , Lazarevic D., Rodriguez J., Schneider C. (1997). Dismantling cell-cell contacts during apoptosis is coupled to a caspase-dependen t proteolytic cleavage of beta-catenin. J Cell Biol139: 759—771.
9.
Cantley LC, Neel BG ( 1999). New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/ AKT pathway . Proc Natl Acad Sci USA96: 4240—4245.
10.
Cardone MH, Roy N., Stennicke HR, Salvesen GS, Franke TF, Stanbridge E., Frisch S., Reed JC ( 1998). Regulation of cell death protease caspase-9 by phosphorylation . Science282: 1318—1321.
11.
Chen J., Cohn JA, Mandel LJ (1995). Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury. Proc Natl Acad Sci USA92: 7495—7499.
12.
Chi XJ, Hiwasa T., Maki M., Sugaya S., Nomura J., Kita K., Suzuki N. (1999). Suppression of okadaic acid-induced apoptosis by overexpression of calpastatin in human UV(r)-1 cells. FEBS Lett459: 391—394.
13.
Crowley E., Horwitz AF (1995). Tyrosine phosphorylation and cytoskeletal tension regulate the release of fibroblast adhesions. J Cell Biol131: 525— 537.
14.
Davis MA, Carbott DE (1999). Herbimycin A and geldanamycin inhibit okadaic acid-induced apoptosis and p38 activation in NRK-52E renal epithelial cells . Toxicol Appl Pharmacol161: 59—74.
15.
Davis MA, Chang SH, Trump BF (1996). Differential sensitivity of normal and H-ras oncogene-transforme d rat kidney epithelial cells to okadaic acid-induced apoptosis. Toxicol Appl Pharmacol141: 93—101.
16.
Davis MA, Smith MW, Chang SH, Trump BF (1994). Characterization of a renal epithelial cell model of apoptosi s using okadai c acid and the NRK-52E cell line. Toxicol Pathol22: 595—605.
17.
Eguchi Y., Shimizu S., Tsujimoto Y. (1997). Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res57: 1835—1840.
18.
Everitt JI, Goldsworthy TL, Wolf DC, Walker CL (1992). Hereditary renal cell carcinoma in the Eker rat: A rodent familial cancer syndrome. J Urol148: 1932—1936.
19.
Everitt JI, Goldsworthy TL, Wolf DC, Walker CL (1995). Hereditary renal cell carcinoma in the Eker rat: A unique animal model for the study of cancer susceptibility. Toxicol Lett82—83: 621—625.
20.
Fadok VA, de Cathelineau A., Daleke DL, Henson PM, Bratton DL (2001). Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosi s of apoptotic cells by macrophages and fibroblasts. J Biol Chem276: 1071—1077.
21.
Fladmark KE , Brustugun OT, Hovland R., Boe R., Gjertsen BT, Zhivotovsky B., Doskeland SO (1999). Ultrarapid caspase-3 dependent apoptosis induction by serine/threonine phosphatase inhibitors. Cell Death Differ6: 1099— 1108.
22.
Freed JJ, Howard S., Lerro A., Dodson L., Scsok D., Knudson AG (1990). Hereditary renal tumors in the rat: Cell lines from adenocarcinoma s induced by the Eker mutation. Proc Am Assoc Cancer Res31: 317.
23.
Goldstein JC , Waterhouse NJ, Juin P., Evan GI, Green DR (2000). The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant . Nat Cell Biol2: 156—162.
24.
Green AJ, Smith M., Yates JR (1994). Loss of heterozygosity on chromosome 16p13.3 in hamartomas from tuberous sclerosis patients. Nat Genet6: 193—196.
25.
Harrington EO, Smeglin A., Newton J., Ballard G., Rounds S. (2001). Protein tyrosine phosphatase-dependen tproteolysis of focal adhesion complexes in endothelial cell apoptosis. Am J Physiol Lung Cell Mol Physiol280: L342—353.
26.
Henske EP, Neumann HP, Scheithauer BW, Herbst EW, Short MP, Kwiatkowski DJ (1995). Loss of heterozygosity in the tuberous sclerosis (TSC2) region of chromosome band 16p13 occurs in sporadic as well as TSC-associated renal angiomyolipomas. Genes Chromosomes Cancer13: 295—298.
27.
Hino O., Klein-Szanto AJ, Freed JJ, Testa JR, Brown DQ, Vilensky M., Yeung RS, Tartof KD, Knudson AG (1993). Spontaneous and radiation-induced renal tumors in the Eker rat model of dominantly inherited cancer. Proc Natl Acad Sci USA90: 327—331.
28.
Howe SR, Gottardis MM, Everitt JI, Goldsworthy TL, Wolf DC, Walker C. (1995). Rodent model of reproductive tract leiomyomata. Establishment and characterization of tumor-derived cell lines. Am J Pathol146: 1568—1579.
29.
King MA, Radicchi-Mastroianni MA, Wells JV (2000). There is substantial nuclear and cellular disintegration before detectable phosphatidylserine exposure during the camptothecin-induce dapoptosis of HL-60 cells. Cytometry40: 10—18.
30.
Kondo T., Takeuchi K., Doi Y., Yonemura S., Nagata S., Tsukita S. (1997). ERM (ezrin/radixin/moesin )-based molecular mechanism of microvillar breakdown at an early stage of apoptosis. J Cell Biol139: 749—758.
31.
Krautheim A. , Brechlin P., Becker K., Winkler M., Steinfelder HJ (2000). Hamster pancreatic beta cell lines with altered sensitivity towards apoptotic signalling by phosphatase inhibitors. Br J Pharmacol129: 687—694.
32.
Kubo Y., Klimek F., Kikuchi Y., Bannasch P., Hino O. ( 1995). Early detection of Knudson's two-hits in preneoplastic renal cells of the Eker rat model by the laser microdissection procedure. Cancer Res55: 989—990.
33.
Kubo Y., Mitani H., Hino O. ( 1994). Allelic loss at the predisposing gene locus in spontaneous and chemically induced renal cell carcinomas in the Eker rat. Cancer Res54: 2633—2635.
34.
Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227: 680—685.
35.
Leist M., Single B., Castoldi AF, Kuhnle S., Nicotera P. (1997). Intracellular adenosine triphosphate (ATP) concentration: A switch in the decision between apoptosis and necrosis. J Exp Med185: 1481—1486.
Mukerjee N., McGinnis KM, Park YH, Gnegy ME, Wang KK ( 2000). Caspase-mediated proteolytic activation of calcineurin in thapsigarginmediated apoptosis in SH-SY5Y neuroblastoma cells. Arch Biochem Biophys379: 337—343.
38.
Orimoto K., Tsuchiya H., Sakurai J., Nishizawa M., Hino O. ( 1998). Identification of cDNAs induced by the tumor suppressor Tsc2 gene using a conditional expression system in Tsc2 mutant (Eker) rat renal carcinoma cells. Biochem Biophys Res Commun247: 728—733.
39.
Rossini GP, Sgarbi N., Malaguti C. (2001). The toxic responses induced by okadaic acid involve processing of multiple caspase isoforms. Toxicon39: 763—770.
40.
Rytomaa M., Lehmann K., Downward J. (2000). Matrix detachment induces caspase-dependent cytochrome c release from mitochondria: Inhibition by PKB/Akt but not Raf signalling . Oncogene19: 4461—4468.
41.
. Santoro MF, Annand RR, Robertson MM, Peng YW, Brady MJ, Mankovich JA, Hackett MC, Ghayur T., Walter G., Wong WW, Giegel DA (1998). Regulation of protein phosphatas e 2A activity by caspase- 3 during apoptosis. J Biol Chem273: 13119—13128.
42.
Spinedi A., Di Bartolomeo S., Di Sano F., Rodolfo C., Ambrosino A., Piacentini M. (1999). Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatas e inhibitor okadaic acid. Cell Death Differ6: 618—623.
43.
Suganuma M. , Fujiki H., Suguri H., Yoshizawa S., Hirota M., Nakayasu M., Ojika M., Wakamatsu K., Yamada K., Sugimura T. (1988). Okadaic acid: An additional non-phorbol-12-tetradecanoate-13-acetate - type tumor promoter. Proc Natl Acad Sci USA85: 1768 — 1771.
44.
Suuronen T. , Kolehmainen P., Salminen A. (2000). Protective effect of L-deprenyl against apoptosis induced by okadaic acid in cultured neuronal cells. Biochem Pharmacol59: 1589—1595.
45.
Turowski P. , Favre B., Campbell KS, Lamb NJ, Hemmings BA (1997). Modulation of the enzymatic properties of protein phosphatase 2A catalytic subunit by the recombinant 65-kDa regulatory subunit PR65alpha. Eur J Biochem248: 200—208.
46.
van de Water B., Nagelkerke JF, Stevens JL (1999). Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells. J Biol Chem274: 13328—13337.
47.
van de Water B., Tijdens IB, Verbrugge A., Huigsloot M., Dihal AA, Stevens JL, Jaken S., Mulder GJ (2000). Cleavage of the actin-capping protein alphaadducin at Asp-Asp-Ser- Asp633-Ala by caspase-3 is preceded by its phosphorylation on serine 726 in cisplatin-induced apoptosis of renal epithelial cells. J Biol Chem275: 25805—25813.
48.
Walker C., Recio L., Funaki K., Everitt J. (1992). Cytogenetic and molecular correlates between rodent and human renal cell carcinoma. Prog Clin Biol Res376: 289—302.
49.
Zhan Y., van de Water B., Wang Y., Stevens JL (1999). The roles of caspase-3 and bcl-2 in chemically-induce d apoptosis but not necrosis of renal epithelial cells. Oncogene18: 6505—6512.
50.
Zhuang J., Dinsdale D., Cohen GM (1998). Apoptosis, in human monocytic THP.1 cells, results in the release of cytochrome c from mitochondri a prior to their ultracondensation, formation of outer membrane discontinuities and reduction in inner membrane potential. Cell Death Differ5: 953—962.
