10x4 C h t k a t o g r . 430.249- 26 2 82. Desser. H., Kleinberger, G. & Klaring, J. ( I98 1 ) J. Clin. (’hem. C’lin.Biochem. 19, 1 59- 164 83. Desser, H., Frass, M., Kumits, R., Aiginger, P., Muller, M. M. & Klaring, W. J. ( 198 I ) Adv. I’olyarnine Res. 3 , 4 3 1-440 84. Colombatto, S., Fasulo, L., Fulgosi, B. & Grillo, M. A. ( 1 990) In!. J. Hiochem. 22,489-492 85. Kakinuma, Y.,Hosino, K. & Igarashi, K. (1988) Eur. J. niochem. 176,409-41 4 86. Moulinoux. J.-Ph., Quemener, V., Khan, N. A,. Delcros. J.-G. & Havouis, R. ( 1989)Anticancer Res. 9, 1057- I062 87. Moulinoux, J.-Ph., Quemener, V., Khan, N. A,, Havouis, R. & Martin, C. ( 1989)Anticancer Res. 9, 1063- 1068 88. Bethell, 0. R. & P e g , A. E. (1981) J. Cell. I’hysiol. 109, 46 1-468 89. Van Den Bosch, L., De Smedt, H., Missiaen, L., Parys, J. B. & Borghgraef, R. ( 1990) Biochem. J. 265,609-6 12 90. Martin, R. L., Ilett, K. F. & Minchin, R. F. (1990) Biochim. Hiophys. Actu 1051,52-59 91. Byers, T. L. & Pegg, A. E. (1989) A m . J. I’hysiol. 257, cs45-cs53 92. Kumagai, J., Jain, K. & Johnson, L. R. (1989) A m . J. I’hysiol. 256, G90S-G910 93. Rannels, D. E., Kameji, R., P e g , A. E. & Rannels, S. R. (1989) A m . J. I’hysiol. 257, L346-L353 94. Saunderb, N. A,, Ilett, K. F. & Minchin, R. F. (1989) J. C ’ d . I’hysiol. 139,624-63 I 95. Maekawa, S., Hibasami, H., Uji, Y.& Nakashima, K. (1989) Riochim. Biophys. A m 993, 199-203 96. McCormack, S. A. & Johnson, L. R. (1989) A m . J. I’hysiol. 256, G868-G877 97. Heston, W. D. W., Watanabe, K. A,, Pankiewicz, K. W. & Covey, D. F. ( 1987) Biochem. I’hrirmacol. 36, 1849- 1852 98. Alhonen-Hongisto, L., Seppanen, P. & Janne, J. (1980) Niochem. J. 192,941-945 99. Minchin, R. F., Martin, R. L., Summers, L. A. & Ilett, K. F.

BlOCHEMICAL SOCIETY TRANSACTIONS (1989)Biochem. J. 262,391-395 100. Persson, I., Khumutov, A. R. & Khumutov, R. M. (1989)

Biochem. J. 257,929-931 101. Kremmer, T., Palyi, I., Holczinger, L., Lorincz, I.,Boldizsar, M. & Paulik, E. ( 1988) Exp. Cell. Biol. 56, 13 I - 137 102. Niskanen, E., Kallio, A,, McCann, P. P. & Baker, I). G. ( 1983) Blood61,740-745 103. Benalal, D. & Bachrach. U. ( 1 9 8 5 ) Biochem. J. 227,389-395 104. Rinehart, C. A. & Chen, K. Y. ( I 984) J. Biol. (’hem. 259, 4750-4756 105. Poulin, R., Secrist, J. A. & Pegg, A. E. ( 1989)J. Riochem. 263, 21 5-221 106. Porter, C. W., McManis, J., Casero, R. A. & Rergeron, R. J. ( 1987) Cancer Res. 47,282 1-2825 107. Kramer, D. L., Khomutov, R. M., Bukin, Y. V., Khomutov, A. R. & Porter, C. W. ( 1 989) Biochem. J. 259,325-331 108. Steglich, C. & Scheffler, 1. E. (1982) J. Hiol. C’hem. 257. 4603-4609 109. Anehus, S., Pohjanpelto. P., Baldetorp. B., Langstrom. E. & Heby, 0.( 1984) Mol. Cell. B i d . 4.9 15-922 1 10. Van Den Berg, G., Kingma, A. W. & Muskiet, F.A. J. ( 1987)J. Chromatogr. 415,27-34 1 1 I . Elworthv, P. & Hitchcock, E. (1989) Riochim. Hiophys. . . Acta 993.2 1 i2-2 126 1 12. Canellakis. Z. N.. March. L. L.. Young. ”, P. & Bondv. P. K. ( 1984) Cancer Res. 44,3841 -3845 1 1 3. Bethell, D. R., Hibasami, H. & Pegg, A. E. (1982) Am. J. I’hySiol. 2 43. C26 2 -C26 9 1 14. Pakala, R.,Laskov, R., Rottem, S. & Bachrach, U. ( I 988) FEMS Microbiol. Lett. 49, 357-36 I I IS. Pegg, A. E., Tang. K.-C. & Coward, J. K. ( 1982) 8iocherni.stry 21.5082-5089

Received 18 June I990

Polyamine-mediated control of ornithine decarboxylase and S-adenosylmethionine decarboxylase expression in mammalian cells OLLE HEBY,* INGVAR HOLM* and LO PERSSONt * Ilepurtmetit of Zoophysiology, Utiiversity of Umed, S-WIN7 Utned, Sweden urid tllepurtmetit ~ f I ’ h y . ~ i o l qUniversity y, of Lirrrd, S-22.3 62 Liitid, Swedeti The polyamines putrescine, spermidine and spermine are intracellular regulators o f cell growth and differentiation [ I , 21. As a result of polyamine depletion, cells cease to grow and proliferate, but, with the exception of certain cell types, they d o not die. Thus, polyamine replenishment usually causes resumption of a normal growth rate. Inhibitors of the rate-limiting enzymes in polyamine synthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase ( AdoMetDC), in addition to being antiproliferative in action, induce terminal differentiation of certain tumours of embryonal origin [3]and exert a strong antiparasitic effect [ 4 I. The parasites affected include Tryputio~ornu hrircei gumhierise, which causes African sleeping sickness, and I’tieirmocyvsris curirzii, which is an opportunistic pathogen in AIDS patients and in other immunocompromised hosts. The intracellular polyamine levels are finely tuned by a great variety o f regulatory mechanisms [ I , 21. There is evidence for transcriptional, post-transcriptional, translational and post-translational controls of the polyamine biosynthetic enzymes. Moreover, the polyamine levels are regulated by catabolic reactions and by transport into and out o f the cells. The present paper analyses in particular Abbreviations used: ODC. ornithine decarboxylase; AdoMct DC, S-adcnosylmethionine decarboxylasc; IIFMO. a-difluoromethylornithine; ORF. open reading frame.

those regulatory steps in ODC and AdoMetDC expression which are governed by polyamines. Mouse cells have been shown t o contain a family o f ODC genes. In cells resistant to u-difluoromethylornithine (DFMO),an enzyme-activated irreversible inhibitor of ODC, one of the ODC genes is greatly amplified, indicating that this particular gene represents a functional one [ 51.The other ODC-related genes are believed t o be pseudogenes. The ODC gene of yeast and trypanosome lacks introns 161, whereas mammalian ODC genes are interrupted by 1 1 introns (Table 1 ) [7-121. The twelfth exon o f the murine ODC gene terminates at either of two sets o f the strongly conserved consensus sequence (AATAAA) [ 1 0 , 131, which serves as a polyadenylation signal. This finding explains the two mRNA species observed in murine cells. The upstream region of the mammalian O D C gene is extremely GC-rich and contains a TATA box, a CAMP-responsive element, a CAAT box, a G C box and an AP-2 binding site 17-12]. The first intron, which is more than 2 kb long, contains two putative SPI binding sites, two G C boxes and an AP-1 binding site in the opposite strand 191. The human ODC gene is considerably longer than the murine counterpart, partly because of Ahr repeats in some of its introns 191. Notably, the mouse ODC promoter region is about as strong as that of the Rous sarcoma virus long terminal repeat, and somewhat stronger than the SV40 promoter 171. The haploid human genome seems to contain only a single O D C gene. This is consistent with the presence of thc one ODC mRNA species in human cells. In mammalian ODC mRNAs (Tablc 2 ) [ 8, 10, 13- 17 I the open reading frame ( O R F ) that encodes the ODC protein is

1085

POLYAMINES Table 1. Characteristics of the mammalian ODCand AdoMetllC'genes ODC

Ref.

7 940- 7 946 6514 6509

[9,11l [ l o , 12) [81

2p23ter Multigene family 12; multigene family* 7

[35,36] [ I 01 (361 I371 [n- 121

AdoMetDC

Ref.

6 and X Multigene family

[23)

Size (bp) Human Rat Mouse

Chromosomal localization Human Rat Mouse Hamster Exons

12

[381

Multiple

*ODC-coding sequences have been observed o n at least seven autosomes and on the X chromosome. but these may represent pseudogenes.

Table 2. Characteristics of the mammalinn ODCnnd AdoMetllC' mKNAs ~

ODC

Ref.

AdoMetDC

Ref.

Size (kb) * Human Bovine Rat Mouse Hamster 5' leader (ni) Human Rat Mouse Hamster

2.2-2.3 2.3 2.2 and 2.6 2.0-2.4 and 2.6-2.7 2.2 and 2.7

19, 151 [391 [ 101 [S, 191 [261

333 303-304t 310-313t 284

2.1 and 3.6 2.3 and 3.6 2.1 and 3.4 2.1 and 3.4 2.1 and 3.4

Approx. 320

O H F (nt) Human Rat Mouse Hamster

3' tail (nt) Human Rat Mouse

1383 1383 I383

114,191

1365

[If51

346 3 10 or 697 331 o r 753

19, 151 110, 121

1002 999

557 or I .8- 1.9 kb: 557 or 1.8- 1.9 kb+

'Sizes measured in Northern lots, i.e. including a poly(A) tail in addition to the 3' tail sequenced. t T h e 5' leader o f ODC mRNA contains a small ORF (33nt), with the potential o f encoding a 10 amino acid peptide. However, there is no evidence that it is translated. $The 3' tail of AdoMetDC mRNA contains an ORF, with the potential of encoding a protein. 124 and 125 amino acids long in man and rat, respectively. However, there is n o evidence that it is translated. nt = nucleotides.

preceded by a long leader that is extremely GC-rich (particularly in its 5' portion) and may form secondary structures with very high free stabilizing energy [8, lo]. Notably, this region contains a small ORF, but since the nucleotides flanking its AUG codon d o not perfectly conform to Kozaks consensus sequence [ 181, the translation of this ORF seems rather unlikely [ 141. Human cells have a single ODC mRNA [ 151, whereas murine cells have two, varying in length of the 3' untranslated tail as a result of the two polyadenylation signals in the gene [ 131. The latter species of ODC mRNA are differently expressed among rodents; the smallest one dominates in the mouse [S] whereas the opposite is true in the Chinese hamster [ 171. Mammalian ODCs are composed of two identical subunits, each containing 461 amino acid residues (Table 3) (8-10, 14, 15).The amino acid sequence is well conserved among species [9]. Within the subunit there are 12 cysteine

Vol. 18

residues [ 191, which may explain why ODC is dependent on high thiol concentrations for its activity. Another salient feature of mammalian ODC is its rapid intracellular turnover rate, which may be due to the presence of a PEST region in the C-terminal end [20]. Accordingly, when mouse ODC is truncated in its C-terminal end, the turnover of the enzyme is markedly reduced [ 2 I]. Trypanosome ODC, which is a much more stable protein than the mammalian counterpart, has no corresponding PEST region 161. The slow turnover of ODC in trypanosomes may be the basis for the selective antitrypanosomal action of DFMO. Thus, DFMO may effectively reduce the polyamine level in the parasite, but not in the host in which irreversibly inhibited ODC is continuously being replaced by new active enzyme. Genomic and cDNA clones encoding AdoMetDC have been isolated and sequenced from various eukaryotes (Tables 1 and 2) 122, 231. Apparently, there are multiple

BIOCHEMICAL SOCIETY TRANSACTIONS

1086

Table 3. C'huructeristits qf the mammalian ODC' and AdoMetDC' proteins u s dedutedjkrn rlreir nutleic acid sequences ODC

Size ( k l h ) ,proeri:yrnr Human Rat

Ref.

-

AdoMetDC

Ref.

38.3 (334 aa)

1231 1231

38.1 (333 aa)

Size (klla),uctitte enzyme

Human Kat

Mouse

2 x 5 .2 2 x 5 .o 2 x 5 .2

.Sithunit(s)(ria residues) Human Rat Mouse Hamster

46 1 46 1 46 I 455

PEST region (uu ri~siclicrs) Human Mouse

423-440

il51 1101

( 2 x 7 . 7 ) + ( 2 x 3 0 . 7 ) 1241

[14,101 67 f 266*

1241

242-269

1231

[20]

*The serine residue at position 68 of the AdoMetDC proenzyme is converted to pyruvate 1241. aa =amino acid. genes for AdoMetDC in rodent cells. Whether there is more than one functional gene remains to be determined. In mammals. there are two AdoMetDC mRNA species with different sizes, apparently due to differential utilization of two polyadenylation signals in the same gene. The two species o f mRNA may be differentially expressed. depending on the species, the tissue and the physiological condition. However this phenomenon has not been studied in detail, mainly due t o the low basal level o f the mRNA. Like ODC mRNA, AdoMetDC mRNA has a long 5' leader [23].This leader is also GC-rich, and thus may form strong secondary structures. In addition t o the ORF, which codes for the AdoMetDC enzyme, there is a 372-375 nucleotide ORF in the 3' tail. starting ninc bases after the stop codon 1231. The sequence flanking thc AUG codon in this 3' tail conforms, at least in part, to Kozak's consensus motif [ 181, but whether this ORF is translated remains unknown. At variance with ODC, AdoMctDC is not dcpcndcnt on pyridoxal S'-phosphate, but relies o n covalently bound pyruvate as a prosthetic group. The enzyme is synthesized as an inactive 38 kDa proenzyme, which is cleaved into two polypeptides (Table 3). The site for this cleavage is between glutamic acid 67 and serine 6 8 , generating the pyruvate moiety at the N-terminus of the larger subunit [24], and actual subunit sizes o f 7.7 and 30.7 kDa, respectively. Purified preparations o f mammalian AdoMetDC have only revcaled a 32 kDa subunit on SDS-PAGE. However, using Tricine-SDS-PAGE, it has been shown [23] that the small subunit is indeed precipitated with anti-AdoMetDC antibodies. The small subunit has also been shown to be present in purificd recombinant human AdoMetDC [ 24 1. Analysis of the AdoMctDC cDNA shows that it contains a sequence corresponding t o an amino acid sequence found in pure bovine AdoMetDC [22].The native enzyme appears to contain two small and two large subunits. AdoMetDC, which turns over almost as rapidly as ODC. has a strong PEST region in the large subunit 1231. The importance o f the polyamincs in cell function is reflected in the strict regulatory control of their intracellular concentrations [ 1,2 I. The polyamincs rcgulate their own synthesis by an efficient feedback mechanism [25-3 1 I. When thc endogenous polyamine levels are exhausted, the activities of ODC and AdoMetDC increase. Conversely, when the polyamines arc present in excess. these enzyme activities decrease. The control is exerted mainly at the translational

125-311 and post-translational levels [ 321. The synthesis o f both ODC and AdoMetDC is markedly affected by the cellular polyamine content, without corresponding changes in the ODC and AdoMetDC mRNA Ievels. In fact, the steady-state level of ODC mRNA remains constant even when there is a 10-fold change in the synthesis rate. Evidence for polyamine-mediated translational control has also been obtained in studies of ODC and AdoMetDC translation in reticulocyte lysates [ 28-30]. Changes in the synthesis o f ODC and AdoMetDC promptly affect the amount of the enzymes because o f their extremely rapid turnover rates. The polyamines also regulate the turnover o f ODC and AdoMetDC; putrescine stimulates ODC degradation, and spermidine and spermine stimulate the degradation of both enzymes. The mechanism may involve the synthesis or release of a 22 kDa ODC-inhibitory protein. named antizyme. which binds strongly (but reversibly) to ODC. Binding o f antizyme t o ODC has bccn suggested to mark the enzyme for degradation I33 1. The mechanism behind the polyamine-mediated translational control o f ODC and AdoMetDC is not yet fully understood. The mRNAs for ODC and AdoMetDC belong t o the small class o f mammalian messages that have a 5' leader exceeding 200 nucleotides. The ODC mRNA leader is highly GC-rich and consequently has a very strong tendency to form secondary structures [ 8, 101. This is consistent with the fact that ODC mRNA is poorly translated and usually associated with only a relatively small number o f ribosomes [ 301. It is conceivable that the polyamines, either directly or indirectly. affect the formation and melting o f secondary structures in the 5' leader. and thus thc translation o f the ODC mRNA. The finding that the numbcr o f ribosomes associated with each ODC mRNA molecule is unaffected by the polyamine level indicates that the translational control o f ODC is exerted not only at the level of initiation, but also at the level of elongation [ 301. In addition t o their translational control of AdoMetDC. spermidine and spermine affect the steady-state level of AdoMetDC mRNA [ 3I , 341. Whether this is due t o changes in the rate o f transcription and/or degradation o f the message is presently unknown. Putrescine does not seem to affect the translation o f AdoMet DC. Nevertheless, it regulates AdoMetDC expression by stimulating the cleavage of AdoMetDC proenzyme into the subunits of the active enzyme [ I I. The various mechanisms involved in the regulation o f

1087

POLYAMINES

polyamine s y n t h e s i s are p r e s e n t l y being analysed in f u r t h e r detail, and t h e i n f o r m a t i o n o b t a i n e d may p r o v i d e a basis for the rational development of novel and effective anticancer and antiparasitic agents. T h e authors' laboratories are supported by the Swedish Natural Science and Medical Research Councils, the Swedish Council for Planning and Coordination of Research. the Swedish Cancer Socicty, and the Knut and Alice Wallenberg, the J . C. Kempe. the Magnus Bergvall, and the John and Augusta Persson Foundations.

2 0 . Rogers. S., Wells. R. & Rechsteiner, M. ( 1986) Scicwcv 234. 364-368 2 I , Ghoda. L., van Ilaalen Wetters. rl.. Macrae. M., Ascherman, 1). & Coffino. P. ( 1989)Scieiicc, 243. 1493- 1495 22. Mach, M.. White, M. W., Neubauer. M., Degen, J. L. & Morris, D.R.(1986)J,Hi0/.C'hern. 261. 11697-11703 23, pajuncn, A,, crozat, A,, ~ a 0, A,, ~ ~ h~ ~ ~lR,,~~ , i ~~ ~i ~~ , i P. H., Stanley. B.. Madhubala. R. & Pegg, A. E. (1988)J. Hiol. C'hm~.263, 17040-17049 ~ , J , H ; ( ~ / ~'h~,,,,. , 24, stanley, B, A,, pegg, A , E, ~ ~ ) 1, l(19x9) 264. 1073-2 -. . ,2 _ ~ - - .I079 25. Kahana. C. & Nathans. I>. ( I 9 8 5 ) J. Riol. ('lic~ni.260,

I'egg. A. E. (IO88)C i o i c w - fh. 48.759-774 15390- I S3Y3 Heby. 0. & Persson. L. ( 1990) Tr(wds Niocheni. S c i . 15. 2 0 . H d t t i . E. & Pohjanpelto. P. ( I9Xh) J. KO/. C % c m 261, 153- I58 9502-9508 Oredsson. S. M.. Billgren. M. & Heby. 0. ( 1985) Eicr. J. Cell 27. Persson, L.. Holm. I. & Heby, 0. ( I 986) /;/i/LS /.c,rr. 205, I~iol.38. 335-343 17s- I7X Ihcchi. C . J. & McCann. P. P. ( 19x7)in hihibiiioii o~I'o/y(cmiiie 9-X. Kameji. T.& Pegg. A. E. ( 19x7)J . Iliol. C'hcvn. 262.24-27 ,W~roho/i.\iii(McCann. P. P., Pegg, A. E. & Sjoerdsma, A,, eds.), 29. Persson, L., Holm, I. & Heby, 0. ( 19x8) J. Hiol. C ' h c w i . 263, pp. 3 17-344.Academic Press, New York 3528-3533 McConlogue. L., Gupta. M.. Wu. L. & Coffino. P. ( 1984)/'roc. 30. Holm, I . , Perswn, L., Stjernborg, L., Thorsson, L. & Heby, 0. N(/rl./t(.ti(l. S C ~ .U..S.A.81.540-534 ( I Y X Y j N i o ~ h c ~ t1. i ~258. . 343-350 Phillips. M. A,. Coffino. P. & Wang. C. C. ( I9871 J. H i d . C ' l i c n i . 31. Perswn, L., Stjernborg. L., Holm, I. & Hchy, 0. (19x9) 262.872 1-8727 Hiochcm. H i o p / i w . Kes. C ' o m r n i r i i . 160. 1 196- I202 13rnhnnt. M.. McConloguc. L.. van Daalen Wetters. '1'. S! 37. van Ilaalen Wetters, T., Macrae, M., I3rabant. M., Sittler. A. & CoTTino. P. [ 1988) \'roc. Nor/. Acc~tl.S c i . U.S.A.85. 2200-9-704 Coflino. P. ( 1989) M o l . C'i4l. H i o l . 9.54x4-5490 Kntz. A. & Kahana. C. ( 1988) J. Hiol. C ' I i c w i . 263.7604-7OOY 3 3 . Hayashi, S. & Canellakis. E. S. ( 1989) in Oriiirhiiie /)cwtrbFitzgernld. M. C. & Flanagnn. M. A . [ I Y89) DNA 8.623-034 o.~y/(c,sc:B i o k i ~ /iir;yiiio/ogy, ~, & ,tlo/cwt/tr (;cvieric,.s ( Hayashi. Wen. L. Huang. J.-K. & 13lackshear. P. J. ( 1989)J . Riol. C ' I r c w i . S., ed.). pp. 47-58,Pergamon Press, New York 264. 90 16-00? I 34. Shirahata. A. k I'egg. A. E. ( 19x6) J. BioL C ' I i c w r . 261, viin Stceg. H.. von Oostrom. C. Th. M.. Martens. J. W. M.. van 13833-13837 Kreijl.