Iron deprivation induces apoptosis in some sensitive cultured tumour cells, while other cells are resistant. In order to elucidate the mechanisms involved in apoptosis induction by iron deprivation, we studied the expression of p53 and the expression of selected p53-regulated genes. To discriminate between changes coupled only with iron deprivation and changes involved in apoptosis induction by iron deprivation, we compared the expression of the genes in sensitive (human Raji, mouse 38C13) versus resistant (human HeLa, mouse EL4) cells under iron deprivation. Iron deprivation was achieved by incubation in a defined iron-free medium. The level of p53 mRNA decreased significantly under iron deprivation in sensitive cells, but it did not change in resistant cells. On the contrary, the level of the p53 protein under iron deprivation was slightly increased in sensitive cells while it was not changed in resistant cells. The activity of p53 was assessed by the expression of selected p53-regulated targets, i.e. p21(WAF1/CIP1) gene, mdm2, bcl-2 and bax. We did not detect any relevant change in mRNA levels as well as in protein levels of these genes under iron deprivation with the exception of p21(WAF1/CIP1). We detected a significant increase in the level of p21 mRNA in both (sensitive and resistant) mouse cell lines tested, however, we did not find any change in both (sensitive and resistant) human cell lines. Moreover, the p21(WAF1/CIP1) protein was accumulated in mouse-sensitive 38C13 cells under iron deprivation while all other cell lines tested, including human-sensitive cell line Raji, did not show any accumulation of p21(WAF1/CIP1) protein. It seems that the p21(WAF1/CIP1) mRNA, as well as protein accumulation, is not specifically coupled with apoptosis induction by iron deprivation and that it is rather cell-line specific. Taken together, we suggest that iron deprivation induces apoptosis at least in some cell types independently of the p53 pathway.

Cell Growth Control Laboratory Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague Czech Republic

See more in PubMed

Abeysinghe RD, Greene BT, Haynes R, Willingham MC, Turner J, Planalp RP, Brechbiel MW, Torti FM, Torti SV (2001) p53‐independent apoptosis mediated by tachpyridine, an anti‐cancer iron chelator. Carcinogenesis 22, 1607. PubMed

Antonsson B, Conti F, Ciavatta A, Montessuit S, Lewis S, Martinou I, Bernasconi L, Bernard A, Mermod JJ, Mazzei G, Maundrell K, Gambale F, Sadoul R, Martinou JC (1997) Inhibition of Bax channel‐forming activity by Bcl‐2. Science 277, 370. PubMed

Bhatia K, Goldschmidts W, Gutierrez. M, Gaidano G, Dalla Favera R, Magrath I (1993) Hemi‐ or homozygosity: a requirement for some but not other p53 mutant proteins to accumulate and exert a pathogenic effect. FASEB J. 7, 951. PubMed

Chen TR (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res. 104, 255. PubMed

Chomczynski P, Sacchi N (1987) Single‐step method of RNA isolation by acid guanidinium thiocyanate‐phenol‐chloroform extraction. Anal. Biochem. 162, 156. PubMed

Darnell G, Richardson DR (1999) The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents III: the effect of the ligands on molecular targets involved in proliferation. Blood 94, 781. PubMed

Donfrancesco A, Deb G, De Sio L, Cozza R, Castellano A (1996) Role of deferoxamine in tumor therapy. Acta Haematol. 95, 66. PubMed

El‐Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B (1993) WAF1, a potential mediator of p53 tumor suppression. Cell. 75, 817. PubMed

Farrell PJ, Allan GJ, Shanahan F, Vousden KH, Crook T (1991) p53 is frequently mutated in Burkitt's lymphoma cell lines. EMBO J. 10, 2879. PubMed PMC

Fiers W, Beyaert R, Declerq W, Vandenabeele P (1999) More than one way to die: apoptosis, necrosis, and reactive oxygen damage. Oncogene 18, 7719. PubMed

Frade R, Gauffre A, Hermann J, Barel M (1992) EBV/C3d receptor (CR2) interacts by its intracytoplasmic carboxy‐terminal domain and two distinct binding sites with the p53 anti‐oncoprotein and the p68 calcium‐binding protein. J. Immunol. 149, 3232. PubMed

Fukuchi K, Tomoyasu S, Watanabe H, Kaetsu S, Tsuruoka N, Gomi K (1995) Iron deprivation results in an increase in p53 expression. Biol. Chem. Hoppe Seyler 376, 627. PubMed

Haldar S, Negrini M, Monne M, Sabbioni S, Croce CM (1994) Down‐regulation of bcl‐2 by p53 in breast cancer cells. Cancer Res. 54, 2095. PubMed

Haupt Y, Rowan S, Shaulian E, Vousden KH, Oren M (1995) Induction of apoptosis in HeLa cells by trans‐activation‐deficient p53. Genes Dev. 9, 2170. PubMed

He M, Rennie PS, Dragowska V, Nelson CC, Jia W (2002) A mutant P53 can activate apoptosis through a mechanism distinct from those induced by wild type P53. FEBS Lett. 517, 151. PubMed

Hoffman B, Liebermann DA (1994) Molecular controls of apoptosis: differentiation/growth arrest primary gene response genes, proto‐oncogenes, and tumor supressor genes as positive and negative modulators. Oncogene 9, 1807. PubMed

Huang C, Zhang Z, Ding M, Li J, Ye J, Leonard SS, Shen HM, Butterworth L, Lu Y, Costa M, Rojanasakul Y, Castranova V, Vallyathan V, Shi X (2000) Vanadate induces p53 transactivation through hydrogen peroxide and causes apoptosis. J. Biol. Chem. 275, 32516. PubMed

Ido Y, Muto N, Inada A, Kohroki J, Mano M, Odani T, Itoh N, Yamamoto K, Tanaka K (1999) Induction of apoptosis by hinokitiol, a potent iron chelator, in teratocarcinoma F9 cells is mediated through the activation of caspase‐3. Cell Prolif. 32, 63. PubMed PMC

Jacobson MD, Weil M, Raff MC (1997) Programmed cell death in animal development. Cell 88, 347. PubMed

Janus F, Albrechtsen N, Dornreiter I, Wiesmuller L, Grosse F, Deppert W (1999) The dual role model for p53 in maintaining genomic integrity. Cell. Mol. Life Sci. 55, 12. PubMed PMC

Jurgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D, Reed JC (1998) Bax directly induces release of cytochrome c from isolated mitochondria. Proc. Natl Acad. Sci. USA 95, 4997. PubMed PMC

Kemp JD (1997) Iron deprivation and cancer: a view beginning with studies of monoclonal antibodies against the transferrin receptor. Histol. Histopathol. 12, 291. PubMed

Kovar J (1988) Growth‐stimulating effect of ferric citrate on hybridoma cells: characterization and relation to transferrin function. Hybridoma 7, 255. PubMed

Kovar J, Franek F (1989) Growth‐stimulating effect of transferrin on a hybridoma cell line: relation to transferrin iron‐transporting function. Exp. Cell Res. 182, 358. PubMed

Kovar J, Stunz. LL, Stewart BC, Kriegerbeckova K, Ashman RF, Kemp JD (1997) Direct evidence that iron deprivation induces apoptosis in murine lymphoma 38C13. Pathobiology 65, 61. PubMed

Kovar J, Valenta T, Stybrova H (2001) Differing sensitivity of tumor cells to apoptosis induced by iron deprivation in vitro . In Vitro Cell Dev. Biol. Anim. 37, 450. PubMed

Krude T (1999) Mimosine arrests proliferating human cells before onset of DNA replication in a dose‐dependent manner. Exp. Cell Res. 247, 148. PubMed

Kyhse‐Andersen J (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. Biochem. Biophys. Meth 10, 203. PubMed

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. PubMed

Levine AJ (1997) p53, the cellular gatekeeper for growth and division. Cell 88, 323. PubMed

Lill R, Diekert K, Kaut A, Lange H, Pelzer W, Prohl C, Kispal G (1999) The essential role of mitochondria in the biogenesis of cellular iron‐sulfur proteins. Biol. Chem. 380, 1157. PubMed

Maclean K, Porter JB (1996) Iron chelators induce apoptosis in thymocytes by mechanisms independent of p53 and DNA synthesis inhibition. European Iron Club Meeting, Jaca; proffered paper.

Marzo I, Brenner C, Zamzami N, Jurgensmeier JM, Susin SA, Vieira HL, Prevost MC, Xie Z, Matsuyama S, Reed JC, Kroemer G (1998) Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281, 2027. PubMed

Miyashita T, Reed JC (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293. PubMed

Nelson WG, Kastan MB (1994) DNA strand breaks: the DNA template alterations that trigger p53‐dependent DNA damage response pathways. Mol. Cell Biol. 14, 1815. PubMed PMC

Pallisgaard N, Clausen N, Schroder H, Hokland P (1999) Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real‐time RT‐PCR exemplified by t (12; 21) TEL‐AML1 fusion transcript. Genes Chromosomes Cancer 26, 355. PubMed

Pollice AA, McCoy JP Jr, Shackney SE, Smith CA, Agarwal J, Burholt DR, Janocko LE, Hornicek FJ, Singh SG, Hartsock RJ (1992) Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins. Cytometry 13, 432. PubMed

Raff M (1998) Cell suicide for beginners. Nature 396, 119. PubMed

Reichard P (1993) From RNA to DNA, why so many ribonucleotide reductases? Science 260, 1773. PubMed

Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM (1990) The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63, 1129. PubMed

Seliger B, Papadileris S, Vogel D, Hess G, Brendel C, Storkel S, Ortel J, Kolbe K, Huber C, Huhn D, Neubauer A (1996) Analysis of the p53 and MDM‐2 gene in acute myeloid leukemia. Eur. J. Haematol. 57, 230. PubMed

Selvakumaran M, Lin HK, Miyashita T, Wang HG, Krajewski S, Reed JC, Hoffman B, Liebermann D (1994) Immediate early up‐regulation of bax expression by p53 but not TGF beta 1: a paradigm for distinct apoptotic pathways. Oncogene 9, 1791. PubMed

Shi XB, Nesslinger NJ, Deitch AD, Gumerlock PH, Devere White RW (2002) Complex functions of mutant p53 alleles from human prostate cancer. Prostate 51, 59. PubMed

Smith ML, Ford JM, Hollander MC, Bortnick RA, Amundson SA, Seo YR, Deng CX, Hanawalt PC, Fornace AJ Jr (2000) p53‐mediated DNA repair responses to UV radiation: studies of mouse cells lacking p53, 21, and/or gadd45 genes. Mol. Cell Biol. 20, 3705. PubMed PMC

Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76. PubMed

Taetle R, Honeysett JM, Bergeron R (1989) Combination iron depletion therapy. J. Natl Cancer Inst 81, 1229. PubMed

Tanaka H, Arakawa H, Yamaguchi T, Shiraishi K, Fukuda S, Matsui K, Takei Y, Nakamura Y (2000) A ribonucleotide reductase gene involved in a p53‐dependent cell‐cycle checkpoint for DNA damage. Nature 404, 42. PubMed

Taniguchi T, Endo H, Chikatsu N, Uchimaru K, Asano S, Fujita T, Nakahata T, Motokura T (1999) Expression of p21 (Cip1/Waf1/Sdi1) and p27 (Kip1) cyclin‐dependent kinase inhibitors during human hematopoiesis. Blood 93, 4167. PubMed

Thomas DA, Du C, Xu M, Wang X, Ley TJ (2000) DFF45/ICAD can be directly processed by granzyme B during the induction of apoptosis. Immunity 12, 621. PubMed

Thompson CB (1995) Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456. PubMed

Yan Y, Shay JW, Wright WE, Mumby MC (1997) Inhibition of protein phosphatase activity induces p53‐dependent apoptosis in the absence of p53 transactivation. J. Biol. Chem. 272, 15220. PubMed

Yin Y, Solomon G, Deng C, Barrett JC (1999) Differential regulation of p21 by p53 and Rb in cellular response to oxidative stress. Mol. Carcinog. 24, 15. PubMed

Duodenal expression of iron transport molecules in patients with hereditary hemochromatosis or iron deficiency

Differing expression of genes involved in non-transferrin iron transport across plasma membrane in various cell types under iron deficiency and excess

VDAC2 and aldolase A identified as membrane proteins of K562 cells with increased expression under iron deprivation

Sensitivity of cells to apoptosis induced by iron deprivation can be reversibly changed by iron availability