Polyamine biosynthesis pathway

and Polyamine Interconversions

Arsinsss

S-adenosylmethionine Ornithine **- Arginine

IS-adenosylmethionine j Ornithine C02 ^^Decarboxylase ^ Decarboxylase

Decarboxv-S- Putrescine adenosylmethioninr l / Apao

NH ^SßdbS U,nU»se N'-AcetyJspennidine

Methylthioadenosine v X .-^SSAT

3 \ Spermidine

IgÄ N'-AcetyJsperminc

Spermine S5AT

Fig. 1. Polyamine biosynthesis and catabolism. Spd-S, spermidine synthase, Sp-S, spermine synthase, PAO, polyamine oxidase, SSAT, spermidine/spermine N1 -acetyltransferase.

humans are constantly exposed to environmental carcinogens, and that the human diet contains nitrites and nitrates, which are converted in the gut to potent carcinogen nitrosamines, one may believe that the initiation step of carcinogenesis is inevitable. Furthermore, progression, a critical and late step of carcinogenesis, which involves conversion of a preneoplastic cell to a cancer cell within a benign tumor, involves additional genetic events and is not clearly defined. Thus cancer prevention strategies should be based on knowledge of the mechanism of the promotion step of oncogenesis. The multistep model of mouse skin carcinogenesis has been on the forefront of the identification of irreversible genetic events of initiation and progression, and epige-netic events of tumor promotion (9,10). The type of preneoplastic lesions that develop first in mouse skin with initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-0-tetradecanoylphorbol-13-acetate (TPA) are skin papillomas. The papillomas are benign lesions, some of them regress and others persist and a few develop into invasive squamous cell carcinomas (SCC).

Chronic exposure to ultraviolet radiation (UVR) is the most common etiological factor linked to the development of SCC and basal cell carcinomas (BCC), the most common nonmelanoma forms of human skin cancer. UVA (315-400 nm), UVB (280-315 nm), and UVC (190-280 nm) are the three components of the UV spectrum

Fig. 2. Multistep carcinogenesis.

(11). Since stratospheric ozone absorbs most of the radiation below 310 nm (UVC), the UVR that reaches us on earth comprises mostly UVA (90-99%) and UVB (1-10%). UVA and UVB are the most prominent and ubiquitous carcinogenic wavelengths in our natural environment (11, 12). SCC, unlike BCC, invades the nearby tissues. The first site of metastasis usually is a regional lymph node before metastatic growth in distant sites, such as the lung and brain.

Both TPA and the tumor promotion component of UVR carcinogenesis involve clonal expansion of the initiated cells and result in aberrant expression of genes altered during tumor initiation. TPA and UVR have been reported to alter the expression of genes regulating inflammation, cell growth, and differentiation. Specific examples include upregulation of the expression of p21 (WAF1/C1P1), p53, AP-1 activation, ODC, cyclo-oxygenase-2, tumor necrosis factor (TNF)-a, and a wide variety of cytokines and growth factors (13). Available data indicate that the induction of ODC activity, and the resultant accumulation of putrescine, are essential components of the mechanism of tumor promotion (7).

ODC, which decarboxylates ornithine to putrescine, is characterized by its inducibil-ity and rapid turnover rate (half-life approx 17 min). ODC is the key enzyme in mammalian polyamine biosynthesis. Tumor promoter-induced ODC activity always accompanied increase accumulation of putrescine and spermidine, but spermine levels

TPA DOSE (nmol)

Fig. 3. A correlation between induction of ornithine decarboxylase and squamous cell carcinoma by different tumor promoter TPA doses. Female CD1 mice were initiated with 200 nmol DMBA. Two wk after initiation, all mice were promoted twice weekly with different doses of TPA. Carcinoma incidence was determined at 50 wk of TPA promotion. A separate group of mice were treated with the same doses of TPA, soluble epidermal ODC activity was determined 4.5 h after treatment.

TPA DOSE (nmol)

Fig. 3. A correlation between induction of ornithine decarboxylase and squamous cell carcinoma by different tumor promoter TPA doses. Female CD1 mice were initiated with 200 nmol DMBA. Two wk after initiation, all mice were promoted twice weekly with different doses of TPA. Carcinoma incidence was determined at 50 wk of TPA promotion. A separate group of mice were treated with the same doses of TPA, soluble epidermal ODC activity was determined 4.5 h after treatment.

remained unaltered. The role of ODC has been extensively analyzed in mouse skin tumor promotion by TPA (5-7). Topical application of TPA to the shaved back of a mouse leads to approximately a 200-fold increase in epidermal ODC activity at 4.5 h after treatment. The magnitude of ODC induction is dose dependent and correlates with the ability of the dose to promote skin tumor formation. The degree of induction of ODC activity correlates well with the tumor-promoting ability of a number of structurally unrelated tumor promoters. Furthermore, the ability of a series of phor-bol esters to induce ODC activity correlates with their ability to promote skin tumor formation. Epidermal ODC activity is induced only after treatment of mouse skin with tumor promoters and not after treatment with nonpromoting hyperplastic agents. In contrast, both tumor promoters and hyperplastic agents induce S-ade no sy l -l-methionine decarboxylase activity, an enzyme involved in the biosynthesis of spermidine and spermine. The latter induction may be related to the hyperplasia commonly observed after their applications to mouse skin. An example indicating an association between ODC induction and skin tumor promotion by TPA is illustrated in Fig. 3.

Proportionality between the maximum degree of induction of ODC activity and the number of carcinoma incidence caused by increasing doses of TPA was observed. These findings have led to a conclusion that the induction of mouse epidermal ODC activity may be a rapid screen for new tumor promoters. In fact, Fujiki et al. (14) employed induction of mouse epidermal ODC as one of the rapid tests for the identification of new classes of naturally occurring tumor promoters, such as okadaic acid. A tight link between increased ODC activity and cancer induction prompted the synthesis of an inhibitor of ODC, a-difluoromethylornithine (DFMO).

2.2. DFMO, a Suicide Inhibitor of ODC

DFMO (Fig. 4), a specific irreversible inhibitor of ODC, precludes accumulation of putrescine and spermidine. DFMO is enzymatically decarboxylated and generates an intermediate carbanionic species that, with the loss of fluorine, alkylates a nucleophilic residue at or near the active site, thereby covalently binding the inhibitor to the enzyme.

DFMO is a water-soluble compound and can be administered intraperitoneally or in drinking water. DFMO given in drinking water inhibits the induction of cancers of skin, breast, colon, intestines, and bladder in experimental animals (15,16). The plasma half-life of DFMO in rat, rabbit, dog, monkey, and healthy man has been quantified, and it varies with species from 83 to 353 min. Treatment of animals with high doses of DFMO for a long period results in several side effects, such as suppression of early embryogenesis and arrest of embryonic development, enhanced ovulation in rats, weight loss, diarrhea, thrombocytopenia, and impairment of the development of the brain in preweanling rats. However, such toxic side effects of DFMO were not observed in mice kept for 32 wk on 0.25% DFMO in the drinking water. Toxicity associated with high doses of DFMO in humans is thrombocytopenia and reversible ototoxicity (17,18). DFMO in combination with cyclo-oxygenase-2 inhibitor was a potent therapeutic agent for cancer (19). Aerosol administration of DFMO was effective in the prevention of cancer of the upper respiratory tract of the Syrian golden hamster (20). Also, topical DFMO reduced the number of human actinic keratoses (21).

2.3. To Define a Link Between Polyamines and Cancer Using Transgenic Mouse Models

Several transgenic mouse models overexpressing ODC or polyamine catabolizing enzymes have been generated to link ODC activity and polyamine level to the induction of skin cancer. Halmekyto et al. were first to generate transgenic mice overexpressing the human ODC gene under its own promoter (22). In this ODC-overexpressing transgene, mouse model ODC was overexpressed in all the tissues of the transgenic mice. These ODC-overexpressing transgenic mice elicited enhanced susceptibility to the induction of skin papillomas by the DMBA initiation and TPA promotion protocol (22). Megosh et al. also generated ODC-overexpressing transgenic mice (23), but in these transgenic mice, stable ODC was targeted to the hair follicle using bovine keratin 6 (K6) promoter. A stable ODC expression vector was generated by deleting the PEST sequences from ODC complimentary DNA. As is discussed in Subheading 3, ODC is a labile protein that has a half-life of only 10-20 min. Mammalian ODC is degraded by at least two different pathways comprising constitutive and polyamine-dependent pathways. The PEST sequences (proline [P], glutamic acid [E], serine [S], and threonine [T]) are actually considered as signal structures for rapid and selective degradation of ODC by the proteosome. These K6 ODC transgenic mice spontaneously developed skin papillomas and carcinomas after DMBA initiation or overexpression of v-Ha-ias. T h e s e results indicate that increased ODC activity, as observed in TPA promotion, is sufficient to accomplish the promotion stage of carcinogenesis (24). The enhanced skin tumor development in K6/ODC mice either by DMBA initiation or by photocarcinogenesis was prevented by DFMO (16). These results indicated that increased ODC activity is the key molecule that sensitizes skin for the development of skin tumors.

Further evidence implicating ODC and resultant polyamines in cancer formation emerged from a study involving manipulation of an ODC regulatory protein termed antizyme (AZ) (25-28). AZ is a polyamine-induced cellular protein (molecular weight approx 23 kDa) involved in the feedback regulation of cellular polyamine levels. AZ binds directly to ODC and targets it to rapid ubiquitin-independent degradation by the 26S proteosome. AZ acts on ODC to enhance the association of ODC with proteosome and not the rate of this processing.

There are three isoforms of AZ. ODC AZs 1 and 2 are expressed ubiquitously, and the third, the testicular isoform AZ3, is expressed in differentiated haploid germ cells. AZ1, but not AZ2, accelerates ODC degradation. AZ1 binds ODC with about a threefold higher potency that AZ2, but cannot account for their distinct degradation activities. Evidence also indicates that AZ is not only involved in ODC degradation, but in the negative regulation of polyamine transport. AZ activity is inhibited by the AZ inhibitor, which binds to the AZ with higher affinity than that of ODC, releasing ODC from the ODC-AZ complex (27). AZ is a potential target to regulate cellular polyamine levels and prevent carcinogenesis. AZ overexpression in transgenic mice has been shown to reduce N-nitromethylbenzylamine-induced forestomach carcinogenesis (26). Overexpression of ODC accelerated, but overexpression of AZ inhibited, induction of basal cell carcinogenesis by UVB in PtCh1+/- mice (5).

Further evidence of the role of increase putrescine in skin tumor development was provided using transgenic mice overexpressing spermidine/spermine V1 -ac ety ltransferas e (SSAT). SSAT is characterized by its inducibility and rapid turnover rate, and is the rate-limiting enzyme controlling conversion of spermidine and spermine to putrescine (7). Colemen et al. generated SSAT-overexpressing transgenic mice (K6-SSAT) using the bovine K6 promoter. K6-SSAT transgenic mice appeared to be phenotypically normal and developed 10-fold more skin tumors as compared with wild-type mice in response to single DMBA initiation followed by twice weekly application of T PA. Tumor samples from transgenic mice showed elevated levels of SSAT activity and SSAT protein. These results lend support to the conclusion that activation of polyamine catabolism leading to increases in putrescine and N1 -acetylspermidine may be linked to chemically induced mouse skin neoplasia (7). In contrast, Pietila et al. have reported that SSAT overexpressing transgenic mice developed significantly fewer papillomas than their syngenic littermates (29).

3. ODC Regulation

Molecules that signal ODC induction may be possible targets for cancer prevention and treatment. TPA-induced ODC activity is regulated at least in part at the transcriptional level mediated by non-AP-1 transcriptional factors (3 0-32). In this context, the link of c-Myc in the transcriptional regulation of ODC is noteworthy. c-Myc, an oncogenic transcription factor, is involved in the regulation of various cellular functions. ODC

H2N(CH2)3 - c - COOH a - DIFLUOROMETHYLORNITHINE

Fig. 4. Structure of DFMO, a suicide inhibitor of ornithine decarboxylase.

is a transcriptional target for c-Myc. O D C gene has two conserved functional CACGTG-c-myc binding sites in the first intron, the CATGTG motif in the 5$-flanking region. Transactivation of the ODC promoter also requires the dimerization of c-Myc and Max.

3.1. Protein Kinase C, ODC Induction, and SCC

We also determined the initial signal(s) that are linked to ODC induction by TPA. TPA binds specifically with high affinity to its receptor(s) identified as protein kinase C (PKC) (33-35). PKC, which is ubiquitous in eukaryotes, is a major intercellular receptor for the mouse skin tumor promoter, TPA. PKC forms part of the signal trans-duction system involving the turnover of inositol phospholipids and is activated by DAG, which is produced as a consequence of this turnover. On the basis of the structural similarities and cofactor requirements, the PKC isoforms have been grouped into three subfamilies of enzymes (Fig. 5).

The conventional PKCs (a, bI, bll, and g), which are dependent on DAG/TPA, Ca2+, and phosphatidylserine (PS); the novel PKCs (d, e, q and h), which require only PS and DAG/TPA; and the atypical PKCs (z, l, and i), which retain only the PS dependence but have no requirement for DAG/TPA or Ca2+ for activation (PKCm, which is usually classified as an nPKC, is not easily grouped with any of the other iso-forms) (36-38). At least six PKC isoforms (a, d, e, h, z, and m) are expressed in mouse skin. To determine the in vivo functional specificity of PKCa, -d, and -e in TPA-acti-vated PKC signals to ODC induction and skin tumor multiplicity, we generated transgenic mice that expressed T7-epitope-tagged PKCa, -d, or -e in their epidermis. The expression of individual PKC isoforms was directed to the basal cells of the epidermis using a human cytokeratin 14 (K14) promoter (33-35). Overexpression of PKCa did not affect the induction of skin tumors elicited by the initiation (DMBA)-promotion (TPA) protocol (33, 39). However, overexpression of PKCd suppressed the formation of both skin papillomas and carcinomas (34), whereas PKCe transgenic mice developed papilloma-independent metastatic squamous cell carcinomas (Fig. 6) (3 5,40). Inte re stingly, despite the different skin tumor susceptibilities of PKCa, PKCd, and PKCe transgenic mice, PKCd and PKCe, but not PKCa, overexpressing transgenic mice superinduced epidermal ODC activity compared with wild-type littermates after TPA treatment (33).

3.2. PKCd-Induced ODC Activity is not Linked to the Induction of Skin

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