Báo cáo khóa học: Genetic approaches to the cellular functions of polyamines in mammals potx

Mainly, four different approaches have been applied in the genetic engineering of experimental animals: a activation of polyamine biosynthesis through the overexpression of their biosynt

R E V I E W A R T I C L E

Genetic approaches to the cellular functions of polyamines

in mammals

Juhani Ja¨nne, Leena Alhonen, Marko Pietila¨ and Tuomo A Keina¨nen

A.I Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland

The polyamines putrescine, spermidine and spermine are

organic cations shown to participate in a bewildering

num-ber of cellular reactions, yet their exact functions in

inter-mediary metabolism and specific interactions with cellular

components remain largely elusive Pharmacological

inter-ventions have demonstrated convincingly that a steady

supply of these compounds is a prerequisite for cell

prolif-eration to occur The last decade has witnessed the

appear-ance of a substantial number of studies, in which genetic

engineering of polyamine metabolism in transgenic rodents

has been employed to unravel their cellular functions

Transgenic activation of polyamine biosynthesis through an

overexpression of their biosynthetic enzymes has assigned

specific roles for these compounds in spermatogenesis, skin

physiology, promotion of tumorigenesis and organ

hyper-trophy as well as neuronal protection Transgenic

activa-tion of polyamine catabolism not only profoundly disturbs

polyamine homeostasis in most tissues, but also creates a complexphenotype affecting skin, female fertility, fat depots, pancreatic integrity and regenerative growth Transgenic expression of ornithine decarboxylase antizyme has sugges-ted that this unique protein may act as a general tumor suppressor Homozygous deficiency of the key biosynthetic enzymes of the polyamines, ornithine and S-adenosyl-methionine decarboxylase, as achieved through targeted disruption of their genes, is not compatible with murine embryogenesis Finally, the first reports of human diseases apparently caused by mutations or rearrangements of the genes involved in polyamine metabolism have appeared Keywords: antizyme; ornithine decarboxylase; putrescine; spermidine/spermine N1-acetyltransferase; spermidine; sper-mine; transgenic mouse; transgenic rat

Introduction

The cellular functions of the natural polyamines (putrescine,

spermidine and spermine) are still largely unknown,

although a vast number of studies have shown that these

polycationic compounds are crucial to the growth and

proliferation of mammalian cells Pharmacological

approa-ches are applied typically in studies aimed to unravel their

functions in cellular metabolism and, admittedly, much

valuable information has been generated with the use of

specific inhibitors of polyamine biosynthesis However, the

last decade has produced a substantial number of

experi-mental studies in which genetic engineering of polyamine

metabolism has been used as a tool to elucidate their cellular functions Studies with genetically engineered mice and rats have not only brought entirely new information about the involvement of polyamines in various physiological and pathophysiological processes but they have likewise chal-lenged some of the conventional wisdoms Mainly, four different approaches have been applied in the genetic engineering of experimental animals: (a) activation of polyamine biosynthesis through the overexpression of their biosynthetic enzymes; (b) activation of polyamine catabol-ism through the overexpression of the enzymes involved

in their catabolism; (c) transgenic expression of ornithine decarboxylase (ODC) antizyme, a protein inhibiting ODC activity and facilitating its degradation and (d) gene-disruption technology applied both to the biosynthetic and catabolic enzymes

Polyamine metabolism

Figure 1 outlines the metabolism of the polyamines in a mammalian cell Two amino acids, L-ornithine and

L-methionine, are the primary precursors of the poly-amines L-Ornithine is cleaved from L-arginine by mito-chondrial arginase II [1] or derived from the diet and

L-methionine is first converted to S-adenosyl-L-methionine (AdoMet) Both ornithine and AdoMet are subsequently decarboxylated by two cytosolic decarboxylases, namely ornithine decarboxylase (ODC) and AdoMet decarboxy-lase (AdoMetDC) The former reaction yields putrescine

Correspondence to J Ja¨nne, A.I Virtanen Institute for Molecular

Sciences, University of Kuopio, PO Box1627, FIN-70211, Kuopio,

Finland Fax: + 358 17 163025, Tel.: + 358 17 163049,

E-mail: Juhani.Janne@uku.fi

Abbreviations: ODC, ornithine decarboxylase; AdoMetDC,

S-sylmethionine decarboxylase; dcAdoMet, decarboxylated

adeno-sylmethionine; SSAT, spermidine/spermine N 1 -acetyltransferase;

PAO, polyamine oxidase; SMO, spermine oxidase; DENSPM,

N1,N11-diethylnorspermine; DFMO, 2-difluoromethylornithine;

MGBG, methylglyoxal bis(guanylhydrazone); NMDA,

N-methyl-D -aspartate; GABA, c-aminobutyric acid; eIF5A, eukaryotic

initiation factor 5 A.

(Received 12 December 2003, revised 19 January 2004,

accepted 22 January 2004)

(NH2CH2CH2CH2CH2NH2) and the latter reaction

decarboxylated AdoMet (dcAdoMet) DcAdoMet

do-nates its aminopropyl group either to putrescine in a

reaction catalyzed by a transferase, spermidine synthase

to yield spermidine (NH2CH2CH2CH2NHCH2CH2CH2

CH2NH2), or to spermidine catalyzed by a separate

transferase, spermine synthase to yield spermine

(NH2CH2CH2CH2NHCH2CH2CH2CH2NHCH2CH2

CH2NH2) As the decarboxylation and propylamine

transferase reactions are practically irreversible, an

entirely distinct system exists to convert the higher

polyamines back to putrescine This system utilizes two

different enzymes, a cytosolic spermidine/spermine

N1-acetyltransferase (SSAT) [2] and a peroxisomal

flav-oprotein polyamine oxidase (PAO) [3] As PAO strongly

prefers acetylated polyamines as the substrates [3,4],

SSAT is the rate-controlling enzyme in this

backconver-sion pathway [4] As indicated in Fig 1, spermine can be

either monoacetylated or diacetylated [5] by SSAT As

seen, diacetylation of spermine would not require a

re-entry of spermidine back to the peroxisome but

putres-cine would be formed via N1-acetylspermidine from

diacetylated spermine (Fig 1) In addition to putrescine

and spermidine, the PAO reaction also yields hydrogen

peroxide and acetaminopropanal Working with

SSAT-deficient mouse embryonic stem cells, we found that

SSAT is absolutely necessary for the conversion of

spermidine to putrescine while spermine is readily

converted to spermidine in the absence of SSAT [6]

The conversion of spermine back to spermidine in the

absence of SSAT activity is obviously attributable to a recently discovered amine oxidase, which, when first cloned was thought to be PAO [7], but was soon identified as a novel spermine oxidase (SMO) [8] Unlike PAO, SMO strongly prefers spermine as the substrate over its acetylated derivatives and spermidine is not a substrate at all [8,9] Figure 1 likewise highlights an important Ôside trackÕ of spermidine metabolism, namely

it serves as a precursor for hypusine synthesis This unusual amino acid, derived from the aminobutyl moiety

of spermidine, represents an integral part of eukaryotic initiation factor 5 A (eIF5A) [10] that is essential for eukaryotic cell proliferation [11] A further unique protein, ODC antizyme, is intimately involved in the metabolism of the polyamines Antizyme was initially discovered in the late 1970s as a protein inhibitor of ODC [12–14] Subsequently, it became obvious that the antizyme not only inhibited ODC activity but it also facilitated its degradation by targeting ODC to 26S proteasome [15,16] It also appears that the antizyme is responsible for the feedback inhibition of polyamine transport [17] The regulation of ODC antizyme expres-sion is unique as polyamines directly induce ribosomal frameshifting in decoding antizyme resulting in the formation of full-length functional protein [18,19] Recently, a nuclear localization has been described for antizyme (and ODC) during mouse development [20] and antizyme appears to have nuclear export signals [21] Recent studies have likewise indicated that antizyme interacts in the nucleus with the transcription factor

Fig 1 The metabolism of the polyamines ODC, ornithine decarboxylase; Spd, spermidine; AdoMetDC, S-adenosylmethionine decarboxylase; dcAdoMet, decarboxylated AdoMet; Spm, spermine; SSAT, spermidine/spermine N 1 -acetyltransferase; PAO, polyamine oxidase; SMO, spermine oxidase; eIF5A, eukaryotic initiation factor 5 A The superscripts indicate the genetic modification of the genes: TG, transgenic; KO, knockout;

DN, dominant negative mutation.

Smad1 and with HsN3, a proteasome subunit [22] The

complexthen appears to recruit SNIP 1, the repressor of

CBP/p300 [23] The latter apparently implies that the

antizyme has functions beyond the regulation of ODC

activity/turnover and polyamine transport

There are several recent review articles dealing in more

detail with polyamine metabolism, their putative functions

and polyamine-related pharmacological/clinical approaches

[24–27]

Putative cellular functions of the polyamines

Polyamines are organic cations that are positively charged

under physiological conditions Thus, they are expected to

interact with negatively charged molecules, such as nucleic

acids, phospholipids, etc., within the living cells What

makes them different from divalent cations, for instance?

There are two fundamental differences between the natural

polyamines and divalent cations, such as Mg2+and Ca2+

(a) The positive charges in the polyamines are differentially

spaced within a flexible carbon backbone and hence

electrostatic interactions with other cellular components,

most notably with negatively charged macromolecules, in

all likelihood are more flexible than those exerted by

divalent cations (b) As described above, the polyamines

possess extremely sophisticated metabolic machinery for the

regulation and maintenance of their intracellular

homeo-stasis Of course, one can argue that the latter likewise

holds true for divalent cations in terms of differential

cellular compartmentalization There are more than 1500

research reports describing effects exerted by the polyamines

in great diverse experimental systems in vitro If not all, most

of these experiments may not be relevant as regards the true

cellular functions of the polyamines, as no one knows the

concentrations of free polyamines in a living cell Especially

the higher polyamines, spermidine and spermine while

present in millimolar concentrations in the cell are expected

to be tightly bound to negatively charged cellular

compo-nents and structures leaving only an extremely tiny fraction

of them reactive Thus, to conclude anything about the

functions of the polyamines based on biological effects

exerted by them in vitro is a hopeless task without any solid

ground

As regards the physiological functions of the polyamines,

however, experimental evidence exists that assigns specific

roles to the polyamines in general and to individual

polyamines in particular The polyamines, especially

sper-midine and spermine, interact with DNA with reasonable

specificity In fact, they can alter the structure of DNA, such

as B to Z conversion, and are thus likely to affect the

function of DNA [24] An example of an extremely specific

interaction between the higher polyamines and

polynucleo-tides is the ribosomal frameshifting induced by the

poly-amines in decoding of ODC antizyme [18] Polypoly-amines

appear to have an indispensable role in cell proliferation, as

specific inhibition of their biosynthesis invariably halts

the growth of mammalian cells This likewise applies to

polyamine depletion achieved by an activation of their

catabolism The biosynthetic and catabolic enzymes of the

polyamines have become a meaningful target for cancer

chemotherapy However, the impressive results obtained in

cell cultures and even with tumor-bearing animal models

have not fully translated to clinical practice apparently due

to the sophisticated compensatory mechanisms aimed at maintaining polyamine homeostasis [25,26] In fact, poly-amines may have a dual role in cellular functions by promoting cell growth or inducing apoptosis when they occur in excess [24] An inhibition of polyamine biosynthesis has met much greater success in the treatment of certain parasitic diseases, such as African sleeping sickness, under clinical conditions [24] A further function of the poly-amines, specific to spermidine, is the formation of hypusine,

an integral component of the eIF5A [10] As the latter factor

is essential for the proliferation of mammalian cells [11], it

is often difficult to judge whether spermidine depletion-induced growth arrest is attributable to the polyamine itself

or whether it is related to the shortage of hypusine to form a functional translation initiation factor Finally, the poly-amines specifically interact with certain ion channels, such as N-methyl-D-aspartate receptor, inward rectifying potassium channels and voltage-dependent Ca2+ channels [24] As these interactions occur at such low (nanomolar) concen-trations, it is highly likely that they are relevant also under conditions in vivo

Genetic engineering has been directed to almost every single reaction of polyamine metabolism in transgenic animals and embryonic stem cells Individual enzymes include arginase II, ODC, antizyme, AdoMetDC, spermi-dine synthase, spermine synthase and SSAT the genes of which have been either generally or tissue-specifically overexpressed or disrupted The following sections will describe in detail the consequences of activated polyamine biosynthesis, activated polyamine catabolism, antizyme overexpression and disruption of individual enzyme genes

A short recent review also covers some of the transgenic mouse models [28]

Activation of polyamine biosynthesis

in transgenic rodents

Overexpression ofornithine decarboxylase Polyamine homeostasis The first animal with genetically engineered polyamine metabolism was a transgenic mouse overexpressing the human ODC gene under its own promoter [29] The transgene was overexpressed practically

in all tissues of the transgenic mice in a position-independent and gene copy number-dependent fashion [30] While the parenchymal tissues displayed moderately elevated ODC activity, some tissues, such as testis and brain, showed an enzyme activity that was 20–80 times higher than that in the respective tissues of their nontransgenic littermates The high activity of ODC greatly expanded tissue pools of putrescine, especially in testis and brain [31], but, with the possible exception of testis, was not reflected in any alterations of tissue levels of spermidine and spermine [31] The fact that testis and brain showed the most expanded putrescine pools in the transgenic animals may be attribut-able to their less permeattribut-able blood/tissue barriers than in other tissues This apparent block between putrescine and the higher polyamines is especially puzzling as analyses of SSAT and PAO activities and urinary content of the polyamines gave no signs indicative of an activation

of polyamine catabolism [31] or their enhanced urinary

excretion [32] in the transgenic animals An elevation of

tissue contents of L-ornithine through the inhibition of

ornithine transaminase only further expanded putrescine

pools but not those of spermidine or spermine [32],

indicating that the tissue ornithine pool became rate-limiting

under these conditions These results led us to conclude that

one of the major functions of the polyamine homeostatic

system in nondividing (or nonstressed) mammalian cells is

to prevent an excessive accumulation of the higher

poly-amines spermidine and spermine, in fact, a view already

expressed by Davis et al [33] in their review article If

correct, this would require some sort of physical or chemical

sequestration of putrescine under these conditions

Spermatogenesis While establishing the first transgenic

ODC overexpressing transgenic mouse line (UKU2), we

generated a transgenic founder male displaying extremely

high testicular ODC activity This male turned out to be

infertile and subsequent histological examination of testes

revealed greatly reduced amount of germinal epithelium

and total absence of ongoing spermatogenesis resembling a

syndrome causing infertility in man and known as ÔSertoli

cells-only syndromeÕ [29] A closer examination of the

members of the UKU2 line revealed a significant decrease

also in their sperm count [29] Our subsequent studies

indicated that an enhanced testicular ODC activity has

a dual effect on spermatogenesis; a moderately enhanced

putrescine accumulation stimulates mitotic DNA synthesis

while excessive accumulation of the diamine inhibits meiotic

DNA synthesis, particularly upon advancing of age [34],

ultimately leading to male infertility [29] Highly relevant to

these findings is a recent discovery of a testis germ

cell-specific ODC antizyme (antizyme 3) [35] The expression of

this antizyme is strictly restricted to testis and it is expressed

stage-specifically in postmeiotic germ cells, i.e during late

spermatogenesis [35] As indicated by our results with ODC

transgenics, excessive putrescine accumulation during late

spermatogenesis appears to be detrimental to the germ cells

and, hence, antizyme 3 apparently functions to limit any

ODC expression to the early spermatogenesis The

trans-gene-derived ODC seemingly ÔoverwhelmsÕ the normally

occurring antizyme and leads to an excessive accumulation

of putrescine in late-stage germ cells [35,36]

Spontaneous tumorigenesis Elevated ODC activity and

expanded pools of the polyamines are commonly associated

with tumorigenesis and a role of an oncogene-like protein

has been assigned to ODC [26,37] Under cell culture

conditions, forced expression of ODC appears to result in

malignant transformation [37,38] and cells overproducing

ODC are able to form tumors in nude mice [39] We

subjected the ODC-overexpressing transgenic mice to a

long-term survival study in order to assess whether very

high constitutive tissue ODC activity would predispose the

animals to an enhanced general tumorigenesis At two years

of age, these animals still displayed 20–50 times higher tissue

ODC activity than their syngenic littermates, yet

macro-scopic and micromacro-scopic examination of organs did not

reveal any difference between syngenic and transgenic mice

as regards spontaneous tumor incidence [40] These findings

are supported indirectly by results obtained with growth

hormone overexpressing transgenic mice, which, in addition

to constitutively elevated circulating growth hormone levels, showed enhanced ODC activity in liver and some other tissues but did not display any signs of malignant transfor-mation even at advanced age [41] A more recent study with transgenic mice carrying mammary tumor virus long-terminal repeat-driven ODC cDNA suggests an increased incidence of spontaneous tumors in the transgenic animals [42] The tumors included mammary carcinomas, intestinal adenocarcinoma and a vascular liver tumor, however, a direct connection to ODC is questionable as the listed transgenic tissues displayed lower ODC activity than the corresponding tissues of syngenic mice Another strange feature is the fact that among 17 nontransgenic animals, no pathological findings were observed at 2 years of age, which

is close to the end of the life-span of a mouse [42] According

to a recent report, spontaneous tumor incidence in old mice

of CH3 background (used in the cited study) is 40% [43] Skin.Transgenic mice overexpressing human ODC under its own promoter did not show any macroscopic or microscopic skin abnormalities, yet these animals appeared to be more sensitive than their syngenic littermates to developing skin papillomas in response to the two-stage chemical (initiation and promotion) skin tumorigenesis [44] Targeted (using bovine keratin promoter) overexpression of truncated ODC

in the skin of transgenic mice caused a number of phenotypic abnormalities including early and permanent loss of hair, excessive skin wrinkling, development of dermal follicular cysts, enhanced nail growth and spontaneous tumor devel-opment [45] The fact that hair loss was causally related

to ODC overexpression and putrescine accumulation was convincingly proved by experiments showing that early administration of 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC [46], prevented the hair loss and partially normalized skin histology [47] The same authors likewise showed that hair follicle-targeted overexpression of ODCnot only predisposed the transgenic animals to skin tumorigenesis but the tumors developed in response to a single carcinogen application without a subsequent tumor promotion [48] The use of DFMO reversibly blocked the formation of squamous papillomas in the transgenic animals, which led the authors to conclude that polyamines, most notably putrescine, control the development and mainten-ance of neoplastic phenotype [49] The overexpression of ODC appears to co-operate with v-Ha-ras oncogene as doubly transgenic mice carrying both keratin promoter-driven ODC transgene and v-Ha-ras developed spontaneous skin tumors unlike the singly transgenic animals [50] In addition to chemical carcinogenesis, transgenic mice over-expressing ODC under the control of keratin promoter were also sensitized to photocarcinogenesis as indicated by the more rapid development of skin tumors in these animals in comparison with their syngenic littermates [51] As in the case

of chemical carcinogenesis, the development of tumors in response to the ultraviolet radiation was completely preven-ted by DFMO [51]

The role of ODC in skin tumorigenesis has also been approached by generating transgenic mouse line expressing keratin promoter-driven truncated dominant-negative ODC mutant [52] In spite of an inhibition of wild-type ODC expression in short-term experiments, these animals formed

as many tumors as controls in response to the two-stage skin

tumorigenesis protocol A plausible explanation for the

failure to inhibit tumorigenesis is the competition of the

mutant ODC for the binding of the antizyme and releasing

the wild-type enzyme [52]

The mechanisms whereby ODC overexpression enhances

skin tumorigenesis are largely unknown Two recent reports

link ODC overexpression to histone acetylation as both

histone acetyltransferase and deacetylase activities were

elevated in the skin of the ODC transgenic animals and

histones were hyperacetylated in cultured skin cells

over-expressing ODC [53,54] Skin tumors obtained from doubly

transgenic ODC/Ras mice displayed an exceptionally high

histone acetyltransferase activity [54] These changes were

fully reversible by DFMO treatment These findings may

imply that elevated intracellular polyamines can influence

the chromatin organization and possibly alter specific gene

expression to promote tumor progression [54]

As further regards skin, it may be worth of mentioning

that an overexpression of arginase I in the enterocytes of

transgenic mice elicits arginine deficiency that affects skin,

muscle and lymphoid development, however, in the absence

of altered polyamine tissue pools [55]

Central nervous system The role of the polyamines in

normal and pathological brain is not only under active

research but the existing views of their roles are highly

conflicting A vast number of experiments have shown that

brain insults, either physical or chemical, inevitably activate

the biosynthesis of the polyamines through an induction of

ODC and concomitant accumulation of putrescine This

phenomenon is mostly understood in terms that the

induction of ODC and the enhanced accumulation of

putrescine is causally related to the neuronal damage rather

than representing an adaptive response [56,57] Spermidine

and spermine interact as agonists with the

N-methyl-D-aspartate (NMDA) receptor [58,59], a prolonged

activa-tion of which could be responsible for neuronal damage [56]

Unlike the higher polyamines, putrescine is believed to act

as a weak antagonist of the NMDA receptor [59] The

experiments with transgenic mice and rats overexpressing

ODChave generated new information strongly suggesting

that an enhanced accumulation of putrescine in brain is a

neuroprotective measure rather than a cause of neuronal

damage

As indicated earlier, transgenic mice overexpressing ODC

show the greatest expansion of the putrescine pool in brain

and testis In the long-term survival experiment, we

examined the transgenic animals and their syngenic

litter-mates at 2 years of age and found no macroscopic or

microscopic signs of neuronal degeneration in the transgenic

brains [40] This means that life-long constitutive

over-expression of ODC and enhanced accumulation of

putres-cine in the brain is tolerated with no harmful consequences

Consequent experiments with the ODC overexpressing

mice indicated that these animals showed a significantly

elevated seizure threshold to both chemical

(pentylene-tetrazol) and physical (electroshock) stimuli and impaired

performance in spatial learning and memory tests The

elevated seizure threshold was not due to any changes in the

brain levels of the two major neurotransmitter amino acids,

glutamate and c-aminobutyric acid [60] Mg2+ is a well

known voltage-dependent, physiological blocker of the

glutamate-mediated excitatory currents inhibiting ionic conductance through the NMDA channel [61] Thus, elevated free Mg2+ could potentially block the NMDA receptor, yet our studies revealed that free Mg2+ was significantly lowered (40%) in the brain of transgenic animals [62] Taken together, these results suggest that endogenous putrescine may play a physiologically relevant role at the NMDA receptor as these receptors have a well documented role in the induction of seizure activity [63] and mediating spatial encoding [64] The finding indicating that the transcript levels of several neurotrophins were elevated

in the brain of the transgenic animals may likewise contribute to the apparent neuroprotection [65]

The view that elevated brain putrescine offers neuropro-tection, or at least is not neurotoxic, was supported by a series of studies where ODC overexpressing transgenic mice and rats were subjected to cerebral ischemia Transgenic mice suffering from incomplete forebrain ischemia due to the occlusion of common carotid arteries did not show any signs indicative of putrescine neurotoxicity as judged by changes of energy metabolism (assessed with the aid of nuclear magnetic resonance spectroscopy), induction of immediate early genes and the extent of hippocampal necroses [66] Similar results were obtained with a transgenic rat model for ODC overexpression after permanent middle cerebral artery occlusion [67] A comparison of ODC overexpressing transgenic rats with syngenic and DFMO-treated rats after transient middle cerebral occlusion indi-cated that the ischemia-reperfusion damage developed more slowly and the infarct volumes were smaller in the trans-genic animals [68,69] These studies clearly indicate that an induction of ODC and the concomitant accumulation of brain putrescine are adaptive responses to noxious insults and do not enhance the lesion development

Cardiac hypertrophy Agents that cause cardiac hyper-trophy are known to activate polyamine biosynthesis and elevate their cardiac levels [70,71] Although cardiac hyper-trophy in response to b-adrenergic agonists can be prevented

by a specific inhibition of ODC by DFMO [71,72], it is by no means clear whether an enhanced polyamine biosynthesis and accumulation per se can cause cardiac hypertrophy This issue was addressed by generating transgenic mice with targeted overexpression of ODC in the heart Using truncated ODC driven by a-myosin-heavy-chain promoter,

a more than 1000-fold overexpression was achieved [73] Cardiac putrescine pool was expanded by a factor of 50 and that of spermidine by a factor of four while spermine content was only slightly elevated [73] The enormous ODC activity apparently depleted tissue ornithine, as substantial amounts

of lysine-derived cadaverine accumulated in the heart Even though the altered polyamine pools in transgenic animals did not lead to hypertrophic phenotype, this condition co-operated with b-adrenergic stimulation resulting in severe, sometimes fatal, cardiac hypertrophy compared with only mild hypertrophy in the similarly treated nontransgenic littermates [73] It is noteworthy that in this study, ODC overexpression resulted in substantial expansion of tissue spermidine pool too that is not often observed in other ODC overexpressing transgenic models

Polyamines are known to be modulators of inward rectifying K channels (K channels) spermine being 100-fold

and spermidine 10-fold more potent blockers of the

channels than putrescine or Mg2+ [74–76] Studies on

inward rectification properties of cardiomyocytes isolated

from transgenic mice with heart-targeted ODC

overexpres-sion, unexpectedly revealed that, in spite of massive

over-accumulation of putrescine and cadaverine, the rectification

properties were essentially unaltered [76] Two explanations

for this finding were offered: (a) these diamines did not

significantly contribute to the rectification or (b) their free

concentrations were not altered despite the massive rise in

total levels Interestingly, the authors reached the conclusion

proposing that most of the putrescine (and cadaverine) is

not free but is sequestered within the cell [76] If correct, this

would also explain the commonly observed biosynthetic

block from putrescine to spermidine under conditions of

massive putrescine over-accumulation

In order to exploit whether the tissue concentrations of

spermidine and spermine could be increased, we generated

several transgenic mouse lines overexpressing rat

Ado-MetDC gene Among the five lines produced, none

displayed an increase in AdoMetDC activity nearly as

dramatic as seen in ODC overexpressing animals The

increase in the enzyme activity was at the best fivefold in

comparison with their syngenic littermates and the tissue

pools of spermidine and spermine of transgenic animals

showed only marginal changes [77] Also, in hybrid mice

overexpressing both ODC and AdoMetDC, the tissue levels

of the higher polyamines did not differ from those in

syngenic mice Pulse labeling experiments with primary fetal

fibroblasts obtained from doubly transgenic mice indicated

that polyamine flow was faster in the transgenic than in

nontransgenic fibroblasts AdoMetDC overexpressing

ani-mal did not show any phenotypic alterations [77]

One transgenic mouse line likewise generated moderately

(two–sixfold) overexpressing human spermidine synthase

gene but showing no perturbations in tissue polyamine

homeostasis or phenotypic changes [78] With combined

overexpression of ODC and spermidine synthase in hybrid

transgenic mice neither brought about any changes in

normal polyamine homeostasis [78]

Activation of polyamine catabolism

Overexpression ofspermidine/spermine

N1-acetyltransferase

Polyamine homeostasis As indicated earlier (Fig 1),

sper-midine and spermine are converted back to putrescine

through the concerted action of SSAT and PAO PAO is

constitutively expressed and strongly prefers acetylated

polyamines as the substrates [3] while SSAT is highly

inducible, has a very short half-life and serves as the

rate-controlling enzyme in polyamine backconversion [4] The

first founder animal overexpressing SSAT (UKU169F0)

was a female mouse harboring more than 50 SSAT gene

copies in its genome The animal was extremely small,

hairless and infertile Tissue polyamine pools were

dramat-ically distorted The transgenic brain contained an extremely

high concentration of N1-acetylspermidine, a compound

not normally found in mouse tissues and greatly reduced

spermidine pool, while in liver, the putrescine pool was

strikingly expanded and that of spermine greatly reduced

[79] As the animal was infertile, no transgenic line could be established The second founder animal was male that gave rise to two different kinds of offspring, animals harboring only a few SSAT gene copies and retaining their hair (line UKU165a) and animals having more than 20 SSAT gene copies and permanently losing their hair at 3–4 weeks of age (line UKU165b) Members of the UKU165a showed only marginal alterations in their tissue polyamine pools while members of the UKU165b displayed polyamine pool changes typical for SSAT overexpression: large increase in tissue putrescine pool, appearance of N1-acetylspermidine and decreases in spermidine and/or spermine pools [79] Interestingly, these changes developed in the presence of only moderately elevated tissue SSAT activity [79] Our results (unpublished) have indicated that overexpression of SSATunder these conditions does not decrease the hepatic pool of acetyl-CoA

In an attempt to correct SSAT-induced perturbations in polyamine homeostasis, we generated a hybrid transgenic mouse line overexpressing both ODC and SSAT under the control of mouse metallothionein I promoter Unexpect-edly, these animals showed much more striking signs of activated hepatic polyamine catabolism than the SSAT overexpressing animals [80] Even under the condition of severe depletion of spermidine and spermine pools, tremen-dously high tissue putrescine was not driven further to replenish the reduced pool of spermidine We understand from these results that catabolism is the overriding control mechanism in polyamine metabolism [80]

Regulation of transgene-derived SSAT by polyamine analogues SSAT is known to be powerfully induced by the higher polyamines and especially by certain polyamine analogues [4] The regulation of SSAT expression by polyamines and their analogues apparently occurs at many levels of gene expression These include enhanced transcrip-tion and stabilizatranscrip-tion of the transcript [81,82], enhanced mRNA translation [83,84] and stabilization of the enzyme protein [85] The transgenic animals and cells derived from them typically accumulate large amounts of SSAT-specific mRNA that is, however, extremely poorly translated in the absence of polyamines or their analogues [79,86] This is exemplified by the observation showing that in the pre-sence of N1,N11-diethylnorspermine (DENSPM), a power-ful inducer of SSAT, nontransgenic cells display SSAT activity 10· higher than that in transgenic cells not exposed

to the analogue but containing 10· more SSAT-specific mRNA [86] In fact, working with transgenic mice overex-pressing SSAT under the control of mouse metallothionein

I promoter, we found striking evidence for a post-tran-scriptional regulation of the transgene expression by DENSPM [87] In spite of the heterologous promoter, hepatic transgene-derived SSAT was stimulated more than

40 000-fold by the analogue with marginal changes of transcript levels [87] We proposed that the polyamine analogue could directly interact with SSAT mRNA and improve the translability of the message It is not excluded that polyamine analogues could alter the splicing of SSAT pre-mRNA as certain viruses appear to induce alternative splicing of the SSAT transcript [88]

Polyamine analogues are potential cancer chemothera-peutic agents and, in fact, DENSPM has undergone clinical

trials [89] The antiproliferative action of the polyamine

analogues is believed to be attributable to an induction of

SSAT activity and subsequent depletion of the higher

polyamines This conclusion is based on comparisons

between the different inductions of SSAT activity by the

analogues and their antiproliferative activity [90–93] The

interpretation of such comparisons between genetically

dissimilar cell lines may, however, be difficult as the

analogues may have multiple sites of action in different cell

lines We approached the issue by isolating fetal fibroblasts

from nontransgenic and SSAT overexpressing mice and

exposing the cells to the analogue We now had a pair of

similar cells differing only in the number of SSAT gene

copies We found that the SSAT overexpressing cells were

much more sensitive to DENSPM-induced growth

inhibi-tion than the nontransgenic cells [86] Transgenic mice

overexpressing SSAT were also more sensitive to the general

toxicity of the polyamine analogue [94] A recent report

indicates that small interfering RNA targeted to SSAT

mRNA not only prevented SSAT induction by DENSPM,

but also prevented apoptosis [95]

As will be shown below, overexpression of SSAT in

transgenic animals not only profoundly altered tissue

polyamine homeostasis, but likewise created a very complex

phenotype affecting skin, fat depots, female fertility,

pan-creas, liver and the central nervous system

Skin As indicated earlier, the first founder animal

permanently lost its hair at an early age, as did the

members of the UKU165b line harboring more than 20

copies of the SSAT gene [79] Paradoxically, the hairless

skin histology was practically identical to that found in

transgenic mice with hair follicle-targeted overexpression

of ODC [45], i.e replacement of hair follicles by large

dermal cysts (apparently filled with keratin) and epidermal utriculi, extensive wrinkling of the skin upon aging and lack of subcutaneous fat depots [79,96] Figure 2 shows a young SSAT transgenic mouse with its syngenic littermate (A) and an old transgenic animal displaying excessive wrinkling of the skin that gives a ÔrhinomouseÕ appearance

to the animal (B) The lower panels in Fig 2 depict the histology of normal skin (C), skin of young (D) and an old (E) transgenic mouse Note that the normal hair follicles (C) are replaced by dermal cysts (D), which become extremely large in old animals (E)

In case of ODC overexpression, the hair loss was attributable to an excessive accumulation of putrescine in the skin, as the loss of hair could be prevented by an early administration of the ODC inhibitor, DFMO [47] Although indirectly proved, over-accumulation of putres-cine is in all likelihood responsible for the hair loss also observed in SSAT overexpressing mice This view is supported by experimental findings indicating that putre-scine was constitutively over-accumulated in the skin of these animals and, while the animals properly completed their first hair-cycle, they failed to commence the second anagen phase due to lack of functional hair follicles Moreover, doubly transgenic mice overexpressing both SSAT and ODC with extremely high levels of putre-scine in the skin displayed distinctly more severe skin histology (significantly larger size of the dermal cysts) than did the singly transgenic mice [96] Transgenic mice overexpressing SSAT under the control of mouse metallothionein I promoter also lost their hair but much later than those overexpressing the gene under its own promoter [87]

When subjected to the two-stage skin tumorigenesis protocol, SSAT overexpressing mice developed significantly

Fig 2 Hairless phenotype of the SSAT overexpressing mouse Young SSAT overexpressing mouse with its syngenic littermate (A) An old SSAT transgenic mouse (B) Histology of normal skin showing intact hair follicles (C) Hair follicles are replaced by dermal cysts in SSAT transgenic mouse (D) In old transgenic mouse, the cysts become larger (E) wrinkling the skin.

fewer papillomas than their syngenic littermates [96] This

may be related to the fact that in the syngenic animals, both

ODC activity and skin spermidine level increased much

more in response to the tumor promoter than in transgenic

mice [96] Coleman et al [97] employed another approach

to study the role SSAT in skin pathophysiology producing,

in fact, opposite results They generated transgenic mice

expressing SSAT cDNA under bovine keratin 6 promoter,

which directs the expression to the keratinocytes of the hair

follicle The animals were phenotypically indistinguishable

from their normal littermates and showed normal

hair-cycle The latter may be attributable to the fact that the low

SSAT activity of skin extracts was not increased in the

transgenic animals [97] These animals, however, were much

more sensitive to two-stage skin tumorigenesis, as judged by

tumor incidence and multiplicity, than their syngenic

littermates and showed distinctly enhanced SSAT activity

and increased putrescine and N1-acetylspermidine level in

the papillomas Interestingly, cysts, derived from dilated

hair follicles, were found in the vicinity of the papillomas but

less abundantly elsewhere [97] The obvious inconsistency

between present [97] and previous observations [96] may be

related to the different levels of SSAT expression, genetic

background or hairlessness However, mice used in our

studies [96] were of a BalbC· DBA/2 background Mice of

DBA/2 are reportedly more sensitive to tumor promotion

than animals from a C57BL/6 background [97] Moreover,

all the existing experimental data seem to indicate that an

activation of polyamine catabolism is more closely related

to antiproliferative action than to growth promotion

Female reproductive organs Histopathological

examina-tion of the SSAT overexpressing mice revealed that of 18

tissues and organs examined only skin and female

repro-ductive tract were affected in the transgenic animals Female

members of the transgenic line UKU165b were infertile,

their uteri were hypoplastic due to a thinner muscular layer

and stromal and glandular development was greatly

reduced Examination of the ovaries revealed the presence

of primary and small secondary follicles, but absence of

larger developing follicles and corpus luteum [79]

Differ-ential display analysis of a gene expression profile of uterus

and ovary indicated that the expression of lipoprotein lipase

and glyceraldehyde-3-phosphate dehydrogenase was

eleva-ted in transgenic animals [98] Both enzymes are involved in

energy metabolism and may have detrimental effects on

myometrium and cell viability when overexpressed [98]

SSAT overexpression was also associated with induced

expression of insulin-like growth factor binding protein-2 in

the uterus and ovary and decreased expression of

insulin-like growth factor binding protein-3 in the uterus These

changes may also contribute to the uterine hypoplasia and

ovarian hypofunction [98] It is interesting to note, that

ODC overexpression leads to male infertility while SSAT

overexpression results in female infertility

Pancreas Pancreas is the richest source of spermidine in

the mammalian body and displays the highest molar ratio of

spermidine to spermine, nearly 10 [99,100] The exact

function of such a high spermidine concentration in the

pancreas is not known, but may be related to the intense

protein synthesis that occurs in this organ Pancreatic

growth appears to be dependent on polyamine biosynthesis,

as DFMO retards the growth of the pancreas [101], but does not inhibit the secretory function of the exocrine part of the organ [102] The cellular functions of the polyamines in the pancreas were approached by generating transgenic rat lines overexpressing SSAT under the control of heavy metal-inducible metallothionein I promoter [103] Although transgenic pancreas displayed all the signs of activated polyamine catabolism, such as massive accumulation of putrescine and appearance N1-acetylspermidine, the levels

of the higher polyamines were relatively well maintained Zinc induction of the promoter resulted in a striking stimulation of the pancreatic SSAT activity in the transgenic animals, but not in the syngenic animals, that was accompanied by an almost total depletion of pancreatic spermidine and spermine and development of histologically verified acute necrotizing pancreatitis [103] The possibility that pancreatitis would have been caused by reactive oxygen species generated by the action of PAO was excluded by experiments in which PAO was specifically inhibited before zinc administration, showing that the latter inhibition did not alleviate, but rather worsened the pancreatitis [103] A further piece of evidence causally relating the development

of pancreatitis to the profoundly depleted pancreatic polyamine pools came from experiments showing that the inflammatory process could be totally prevented, as judged

by histopathology and plasma a-amylase activity, by a prior administration of a-methylspermidine, a metabolically stable spermidine derivative [104] The results indicated that the higher polyamines are required for the maintenance of metabolic and structural integrity of the pancreas Induction

of SSAT as a cause of acute pancreatic inflammation may have wider applications, especially concerning drug-induced pancreatitis Gossypol, a cotton seed-derived male antifer-tility agent [105], is known to induce SSAT expression in canine prostate cells [106] We recently showed that the drug activates polyamine catabolism in the pancreas of normal rats and induces acute pancreatitis through a profound depletion of polyamine in transgenic rats overexpressing SSAT [107] It thus appears meaningful to screen drugs known to induce pancreatitis for their effect on pancreatic polyamine catabolism

Liver.Polyamines are intimately associated with the growth

of mammalian cells One of the first animal models demonstrating this involved regenerating rat liver after partial hepatectomy Partial hepatectomy is known to cause

an early induction of ODC in the regenerating liver remnant [108,109] followed by a sequential accumulation of putres-cine and spermidine with a slight decrease in spermine [110] Even though attempts have been made to pharmacologi-cally inhibit rat liver regeneration through blocking ODC, the results have been conflicting [111,112] Partial hepatec-tomy of transgenic rats expressing metallothionein promo-ter-driven SSAT dramatically induced the enzyme at 24 h after the operation that consequently depleted the hepatic spermidine pool by 80% As judged by a number of proliferation indicators, the transgenic rats failed to initiate liver regeneration in striking contrast to their syngenic littermates [113] Only when hepatic spermidine concentra-tion was increased to the preoperative level (apparently due

to very high ODC activity) at day 3 after the operation, liver

regeneration slowly commenced in the transgenic animals

[113] The view that the delayed initiation of liver

regener-ation in the transgenic animals was causally related to the

depletion of hepatic spermidine and spermine pools was

strongly supported by experiments revealing that

a-methyl-spermidine given prior to the partial hepatectomy fully

restored the regeneration [104]

As indicated earlier, SSAT overexpressing mice were

extremely sensitive to the toxic effects exerted by polyamine

analogues Treatment of transgenic mice overexpressing

SSATunder the control of metallothionein promoter with

DENSPM effectively depleted hepatic polyamine pools and

resulted in marked mortality that was associated with

ultrastructural changes in liver, most notably mitochondrial

swelling [87]

Central nervous system In comparison with the ODC

transgenic mice, overexpression of SSAT resulted in even

greater expansion of putrescine pool in different regions of

the brain In situ hybridization analyses of the transgenic

mice indicated that SSAT was overexpressed in all brain

tissue [114] Some experimental work appears to link

enhanced SSAT activity to neuronal damage This view is

based on the finding indicating that kainate-induced seizure

activity resulted in an early stimulation of SSAT activity in

rat brain [115] Neurotoxicity of kainate is mediated by a

Ca2+-dependent process and the drug is particularly toxic to

pyramidal cells of hippocampal and cortical neurons [116]

Overexpression of SSAT appears to protect the transgenic

animals from kainate-induced toxicity This was manifested

as a substantially reduced (50%) overall mortality of the

transgenic mice, in comparison with their syngenic

litter-mates, in response to high-dose kainate [114] The

trans-genicity likewise offered a distinct neuroprotection exhibited

as a reduced expression of glial fibrillary acidic protein, an

commonly used marker of neuronal injury, and no loss of

hippocampal neurons in response to kainate in the

transgenic animals in comparison with wild-type mice

[114] These results support our earlier view suggesting that

expanded pools of brain putrescine, irrespective whether

derived from ODC or SSAT overexpression, have a distinct

neuroprotective role

The SSAT overexpressing mice likewise showed a

significantly elevated threshold, in comparison with their

syngenic littermates, to pentylenetetrazol-induced seizure

activity involving both tonic and clonic convulsions [117]

Although pentylenetetrazol principally induces epilepsy-like

seizure activity through the inhibition of c-aminobutyric

acid (GABA), the major inhibitory neurotransmitter [118], a

number of reports indicate that antagonists of the NMDA

receptor elevate the seizure threshold to pentylentetrazol

[119,120] Interestingly, the difference of seizure threshold

to pentylenetetrazol between the transgenic and wild-type

animals totally disappeared when ifenprodil, a known

NMDA receptor antagonist acting at the polyamine site

of the receptor [121,122], was administered prior to

pentylenetetrazol [117] In addition to the elevated seizure

threshold, SSAT overexpression likewise protected the

animals from pentylenetetrazol-induced neuron loss in

the hippocampus [117] These results are clearly in line with

the notion that grossly elevated putrescine levels or the

greatly increased (up to 40-fold) molar ratio of putrescine to

the higher polyamines in the transgenic brain creates a partial NMDA receptor blockade [117]

Transgenic expression of ODC antizyme

As mentioned earlier, antizyme not only inhibits ODC activity, but also facilitates proteasomal degradation of ODC protein and represses polyamine transport In fact, antizyme occurs in at least three isoforms (antizyme1–3) [16,123] Unlike antizyme 1, which both inhibits ODC activity and facilitates the degradation of the enzyme protein, antizyme 2 appears to lack the latter function [16] As mentioned earlier, antizyme 3 is only expressed in testis during late spermatogenesis [35] Antizyme was shown

to be translocated into nucleus during embryonic develop-ment [20] and the protein contains two independent nuclear export signals [21] Moreover, antizyme forms a ternary complexwith the transcription factor Smad1 and protea-somal subunit HsN3 that is translocated into nucleus in response to bone morphogenetic protein receptor activation [22] In the nucleus, this complexfurther recruits CBP/p300 repressor SNIP1 and is degraded [23] A recent review [123] also suggests, based on so far unpublished report, that cyclin D1 and its associated kinase cdk4 interact with antizyme and are degraded in proteasome in a antizyme-dependent fashion These findings may indicate that ODC antizyme is

a general targeting protein for proteasomal degradation Some recent observations likewise suggest that antizyme possesses functions unrelated to the polyamine metabolism Antizyme expression is up-regulated in melanoma cells

in response to interleukin-1 [124] and antizyme levels are reduced in certain experimental cancers [125] Antizyme seems to play a specific role in mammalian prostate Spermine has been identified as an endogenous growth inhibitor in human prostate [126] and it inhibits the growth

of poorly metastatic, but not of highly metastatic, rat prostate carcinoma cells [127] The failure of spermine to inhibit the latter cells is believed to be attributable to the inability of the polyamine to induce antizyme in the highly metastatic cells [127] If antizyme has functions beyond the metabolism of the polyamines, especially if it interacts with the key players of the cell cycle control, caution should be exercised in interpreting experimental results showing antizyme-dependent growth inhibition

Targeted antizyme 1 expression has been achieved in several transgenic mouse models The structural part of transgene construct used has been a mutated rat antizyme cDNA where a single nucleotide deletion eliminates the need for frameshifting in translation [128]

Cardiac hypertrophy Two transgenic mouse lines were generated constitutively overexpressing mutated antizyme cDNA under the control

of cardiac a-myosin heavy chain promoter targeting the expression to the heart [128] Even though antizyme effectively inhibited cardiac ODC activity, some residual activity was left and the changes in polyamine pools were small with no changes in cardiac function [128]

A prolonged exposure of syngenic mice to isoprenaline elevated cardiac ODC activity, significantly expanded putrescine and spermidine pools and increased cardiac

growth Identical treatment of transgenic mice did not

activate cardiac polyamine biosynthesis and their tissue

accumulation, but induced similar cardiac hypertrophy as

seen in wild-type mice [128] The result was somewhat

unexpected as earlier studies have indicated that a specific

inhibition of ODC by DFMO can prevent b-adrenergic

agonist-induced cardiac hypertrophy [72,129]

Skin tumorigenesis

Antizymeex pression has also been targeted into skin with

bovine keratin 5 and keratin 6 promoters in several lines of

transgenic mice in order to study the role of ODC in the

two-stage skin tumorigenesis In comparison with syngenic

mice, the transgenic mice displayed greatly reduced

epidermal and dermal ODC activity and spermidine

content in response to tumor promotion [130] All the

transgenic lines showed decreased susceptibility to develop

papillomas in response to the two-stage chemical

carcin-ogenesis protocol [130] Although earlier studies have

convincingly shown that a specific inhibition of ODC by

DFMO inhibits skin tumorigenesis in this model [131,132],

the present approach is more specific as the inhibition of

ODC activity by the antizyme occurs in skin cells and not

all over the body

Gastrointestinal carcinogenesis

Chemical carcinogenesis in the fore-stomach of

zinc-defici-ent mice is another model where the role of ODC has been

studied using targeted expression of the antizyme Antizyme

expression significantly reduced both tumor incidence and

multiplicity in response to N-nitrosomethylbenzylamine,

promoted apoptosis and reduced the expression of cyclin

D1 and cdk4 in the fore-stomach of the transgenic mice

[133] The view that the reduced tumor incidence was related

to ODC inhibition and not to direct effects of antizyme on

some cell cycle regulators [123], was strongly supported by

experiments indicating that inhibition of ODC by DFMO

reduced tumor incidence and promoted apoptosis in a

similar fashion as did transgenic expression of antizyme

[133] Based on these and the skin tumorigenesis studies

[130], the authors propose that antizyme may represent a

tumor suppressor gene

Gene disruption technology applied to the

enzymes of polyamine metabolism

Many of the genes of the polyamine metabolizing enzymes

have been disrupted either in transgenic mice or mouse

embryonic stem cells In addition to targeted disruption of

the genes, there is a X-linked dominant mutation in mice that

involves a genomic deletion containing spermine synthase

gene The following paragraphs list the existing knowledge of

disruption of genes involved in polyamine metabolism

Arginase II

Arginase enzyme, degrading arginine to ornithine and urea,

occurs in two isoforms, cytosolic arginase I, which

partici-pates in the urea cycle, and mitochondrial arginase II, which

apparently is involved in the polyamine synthesis [134]

Mice with a targeted disruption of arginase II gene have been created recently Homozygous arginase II-deficient mice were viable and otherwise indistinguishable from wild-type mice, except for showing significantly elevated plasma arginine levels Polyamine analyses of several tissues (brain, liver, kidney and testis) did not reveal any differences between mutant mice and their wild-type counterparts [135] Although the deficiency in arginase II appears to be a benign trait under normal conditions, it is possible that this deficiency could be deleterious under certain pathophysio-logical conditions

ODC Studies employing inhibitors of polyamine biosynthesis have indicated that an inhibition of ODC will arrest murine embryonic development at the morula-blastocyst stage and an inhibition of AdoMetDC at an even earlier stage [136] Moreover, DFMO induces resorption of murine embryos when given just after the first week of gestation [137,138] Studies with mice harboring a disrup-ted ODC gene have revealed that heterozygous animals were viable and fertile while homozygous embryos underwent implantation and induced maternal deciduali-zation, but failed to develop further This was apparently due to marked apoptosis occurring in the pluripotent cells

of the inner cell mass shown as substantial DNA breakage [139] The fact that ODC-deficient embryos developed to the blastocyst stage, i.e., to more advanced stage than those grown in vitro in the presence of DFMO [136], was

in all likelihood attributable to maternal components [139] Attempts to rescue the embryos through supple-mentation of the pregnant females with putrescine were unsuccessful, apparently due to the toxicity of the diamine (high diamine oxidase activity) As to the reasons for lethality of ODC-deficient embryos, the latter authors offer two possibilities: oxidative DNA damage in the absence of polyamines and inhibition of DNA methyla-tion due to excessive accumulamethyla-tion of decarboxylated AdoMet in the absence of putrescine [139]

Similar ODC gene disruption in the nematode Caenor-habditis elegans results in a virtually normal phenotype when grown in complexmedium [140], but when the ODC-deficient nematodes were transferred into polyamine-free medium they showed a phenotype strongly affecting oogenesis and embryogenesis [141]

AdoMetDC

As in the case of ODC, homozygous AdoMetDC deficiency

is not compatible with murine embryogenesis while hetero-zygous animals were viable, normal and fertile [142] AdoMetDC-deficient embryos developed normally to the blastocyst stage, but died shortly thereafter or during the early stage of gastrulation at the latest They developed distinctly further than did embryos cultured in the presence

of an inhibitor of AdoMetDC, methylglyoxal bis(guanyl-hydrazone) [136] When cultured in vitro, AdoMetDC-defi-cient blastocysts showed an absolute growth requirement for spermidine [142] Unlike ODC-deficient blastocysts [139], AdoMetDC deficiency did not result in DNA fragmentation at the blastocyst stage [142] The mouse