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Abstracted from ref. 42.

Abstracted from ref. 42.

shown in Fig. 2, and support the conclusion that ODC is downregulated as a function of age in apparently normal rectal mucosa. When ODC is downregulated with age, as seen by the decrease in spermidine:spermine ratios shown in Fig. 2, the ability of the ODC inhibitor DFMO to reduce spermidine:spermine ratios from baseline values also decreases.

Age (years)

Fig. 3. Proportionate changes in ratios of spermidine:spermine in colorectal biopsies from patients, comparing values after 4 wk of DFMO treatment to baseline values, by participant age. (Reprinted from ref. 42.)

Age (years)

Fig. 3. Proportionate changes in ratios of spermidine:spermine in colorectal biopsies from patients, comparing values after 4 wk of DFMO treatment to baseline values, by participant age. (Reprinted from ref. 42.)

Results from the de-escalation trial of DFMO doses for 1 mo were subsequently corroborated in a randomized clinical trial of 1-yr treatments with one of three DFMO doses vs placebo (43). Eligibility for the trial included men and women, ages 40-80 yr, who had had an adenomatous polyp removed from their colon within the previous 5 yr before entering the trial. Patients were randomized to DFMO doses of 0.075, 0.20, and 0.40 g/m2 or placebo. As shown in Fig. 4, putrescine and spermidine, but not spermine, were decreased in the apparently normal rectal mucosa of participants in a manner dependent on the oral DFMO dose. Spermidine:spermine ratios in rectal mucosa were also reduced in a DFMO dose-dependent manner in this study.

These studies validate measures of colorectal tissue polyamine contents as surrogate markers of DFMO effect for colon cancer chemoprevention trials. The utility of measurement of polyamine contents as marker of DFMO effect in chemoprevention trials is now being demonstrated for other tissue sites. Alberts and colleagues (44) found that DFMO reduced the number of premalignant actinic keratosis lesions and actinic keratosis spermidine contents in a skin cancer chemoprevention trial. We have found that DFMO treatment for 1 mo was associated with a statistically significant decrease in prostatic spermine contents of men undergoing prostate surgical procedures for several indications. The ability of DFMO to suppress tissue spermine appears

Fig. 4. Polyamine content in rectal mucosal biopsy specimens by different DFMO dosage groups (0.075, 0.20, and 0.40 g/m2/d) over time. Values shown for putrescine, spermidine, and spermine are in mmol/mg protein; Spd:Spm = spermidine:spermine ratio; Prot = protein. p values are given for the test for linear trend. (Reprinted from ref. 43.)

Fig. 4. Polyamine content in rectal mucosal biopsy specimens by different DFMO dosage groups (0.075, 0.20, and 0.40 g/m2/d) over time. Values shown for putrescine, spermidine, and spermine are in mmol/mg protein; Spd:Spm = spermidine:spermine ratio; Prot = protein. p values are given for the test for linear trend. (Reprinted from ref. 43.)

to be unique to the prostate, although our initial study included only a small number of men (27). We have not observed DFMO-related reductions in levels of spermine in other human tissues. In a larger, randomized study soon to be reported, we have corroborated our initial finding of a reduction in prostate spermine by DFMO. The mechanism for the tissue-specific reduction of spermine by DFMO is unknown, but may be related to factors that regulate polyamine pool sizes in various tissues. Human prostate contains dramatically higher levels of spermine, relative to other polyamines, compared with other epithelial tissues. For example, levels of spermine are in the range of 3-8 nmol/mg soluble protein in apparently normal squamous esophagus, small intestine, and colon (10,43). Spermidine:spermine ratios are in the range of 0.2-0.8 in these tissues. In apparently normal prostate, average spermine values exceed 20 nmol/mg soluble protein, and spermidine:spermine ratios are less than 0.05 (27).

9. Genetic Variability and Polyamine Pools

A recent study of genetic variability affecting ODC expression has provided evidence that increased polyamine synthesis has a causative role in human colon cancer, rather than being a purely associative effect (45). A single nucleotide polymorphism (SNP), 316 nucleotides downstream of the ODC transcriptional start site, was found to be functionally significant. This SNP is located between two consensus E-boxes in the promoter region. The transcriptional activator c-MYC and the transcriptional repressor MAD1 bind to these elements and modulate promoter activity. The frequency of this SNP has been measured in several groups of people, including participants in one colon cancer prevention trial. Fifty-five percent of participants in this trial, all of whom previously had a colon polyp, were homozygous G at this locus; 35% were heterozygous G/A, and 10% were homozygous A. The transcriptional repressor Madl, a c-Myc antagonist, selectively repressed ODC transcription in an A allele-specific manner. Finally, it was found that the A allele was associated with a statistically significant reduction in risk of colon polyp recurrence. The functional significance of this and other SNPs in the ODC gene and their potential relationship to risk of colon and other epithelial cancers, are under active investigation by others and us. This SNP has been associated with risk of prostate cancer in a case control study (46). Evaluation of polyamine contents in colon and prostate tissues in participants of our cancer chemo-prevention studies, as a function of ODC genotype, is in progress. Genetic variability affecting polyamine metabolic genes could account for interindividual variations in tissue polyamine contents.

10. Polyamine Pools as Biomarkers for Agents Other Than DFMO

Measurement of tissue polyamine contents may be useful biomarkers for agents other than DFMO. Treatments that induce polyamine acetylation can deplete intra-cellular polyamine contents, since monoacetylspermidine and diacetylspermine are substrates for the diamine exporter (DAX) (47). Polyamine analogs that induce SSAT also deplete intracellular spermidine and spermine under certain conditions (48,49). The nonsteroidal anti-inflammatory drug (NSAID) sulindac, a nonselective inhibitor of both cyclo-oxygenase-1 and -2, is used to treat patients with FAP and is under active investigation as a colon cancer chemoprevention agent in combination with DFMO (38). Gene expression profiling of NSAID-treated cultured cells identified altered levels of a number of genes, including several involved in polyamine metabolism. Sulindac suppressed steady state levels of RNA encoding ODC, and increased the levels of SSAT RNA in both human cells in culture and in mice (50). The mechanism of SSAT induction by sulindac involved the activation of PPARy by the sulfone metabolite of sulindac (16). This metabolite lacks the ability to inhibit cyclo-oxygenase activity, suggesting that the mechanism of PPARy activation occurs via a cyclo-oxygenase-inde-pendent mechanism. Activated PPARy subsequently binds to a response element near the start site of SSAT transcription. Sulindac sulfone depletes polyamine contents in human cells in culture and induces apoptosis. Cells can be rescued from sulindac sul-fone-induced apoptosis by exogenous putrescine. Consistent with these results, sulin-dac reduced polyamine contents in intestinal mucosa of mice, especially in those treated in combination with DFMO (unpublished results, manuscript submitted). Sulindac reduced intestinal tumor number in ApcMin/+ mice in a dose-dependent manner. The suppression of intestinal carcinogenesis by sulindac could be partially reversed by supplementing the drinking water with 1% putrescine. Mice drinking putrescine-supple-mented water displayed increased intestinal putrescine concentrations. Aspirin, another NSAID, also induces SSAT promoter activity and RNA expression and causes a reduction in polyamine contents of human cells in culture (45). These results indicate that several NSAIDs affect polyamine metabolic gene expression and exert at least a part of their inhibitory effects on intestinal carcinogenesis via polyamine-dependent mechanisms. Sulindac and DFMO act additively to suppress the growth of colon cancer cells in culture. In sum, these data support the rationale for combination chemoprevention of epithelial cancers using NSAIDS with DFMO. Target tissue polyamine contents may be useful predictive markers to assess the effects of combination cancer chemopreven-tion regimens using NSAIDS in combination with agents that affect polyamine metabolism, or in combination cancer chemotherapy trials employing certain cytotoxic drugs in combination with polyamine analogs (51).

11. Buccal Mucosal Cells are not Surrogates of Other Intestinal Epithelia

Our studies have validated tissue polyamine contents as predictive markers of DFMO effect. However, obtaining tissue for assessment is often difficult, requiring substantial approvals from regulatory and other agencies. Tissue acquisition procedures often cause patient discomfort and limit the number of patients willing to provide specimens for analysis. Consequently, investigators have considered surrogate tissues that might be easier to access as alternative compartments in which to measure polyamine parameters. One of our earliest studies involved the measurement of ODC enzyme activity and polyamine contents in buccal mucosa cells (52). These cells had the distinct advantage that they could be obtained in relatively large quantities by simply brushing the oral cavity with a soft toothbrush, and collecting cells in an oral wash. ODC enzyme activity and polyamine contents were easily measured using methods described in Subheading 5. We noticed, however, that ODC activities varied dramatically depending on the daily time of collection of cells. Further, putrescine was the predominant polyamine detectable in these cells, whereas spermidine and spermine were the predominant polyamines detected in colorectal mucosal tissue of human volunteers. Buccal mucosa cells are in a cellular compartment that is rapidly turning over. We discovered that the majority of cells collected were not viable, based on assessment by vital dye exclusion methods. Several lines of evidence convinced us that the ODC enzyme activity and polyamine contents measured in our buccal mucosal preparations were predominantly of bacterial origin. Electron microscopy revealed that oral bacteria were attached to the dead buccal mucosal cells. Mouth washes with DFMO failed to influence ODC enzyme activities or polyamine contents, whereas similar washing with antiseptics reduced both ODC enzyme activities and polyamine contents. It was known that bacterial ODC is sufficiently different from mammalian ODC and is not suppressed by DFMO. The inhibition of ODC enzyme activity and polyamine contents by antiseptics in buccal mucosal, but not other human cells, was consistent with a bacterial origin of the polyamine parameters we were measuring. The marked dependence of especially ODC enzyme activity likely reflected stimulation of bacterial growth by dietary intake. Thus we concluded that buccal mucosal cells were not reliable surrogates for comparison of polyamine contents in other intestinal epithelia.

12. Urinary Polyamines as Potential Prognostic and Predictive Markers

Polyamine contents in serum and urine have also been measured as potential prognostic markers of disease or predictive markers of therapeutic interventions. Polyamine contents in serum would derive, as export products, from circulating blood cells and other body tissues. Generally, investigators fail to detect changes in serum polyamine contents that are either prognostic for disease progression or predictive of therapeutic responses. Urinary polyamine contents have been correlated with the presence of several cancers and responses of certain cancers to therapeutic interventions. Early studies inconsistently found increased urinary levels of unmodified and monoacetylated polyamines in cancer patients (35). Assessment of urinary polyamine contents in patients participating in a chemoprevention trial failed to detect any effects of DFMO on either specific polyamine contents or spermidine:spermine ratios (53). Most HPLC methods used in early studies of urinary polyamines required free primary amines for detection. Diacetylspermidine and diacetylspermine were undetectable by these methods. Recently, methods have been developed to detect diacetylated forms of spermidine and spermine. Notably, work by Kawakita and colleagues have found that high levels of diacetylspermine are found in the urine of cancer patients (54-57). Urinary levels of diacetylspermine are a more sensitive marker, compared with carcinoembryonic antigen, of several epithelial cancers. Consideration of basic mechanisms of polyamine export supports the contention that extracellular mono- and diacetylpolyamines would be potential markers of elevated polyamine synthesis. Elevated intracellular polyamine pools activate the expression of SSAT, both transcriptionally and posttranscriptionally. SSAT will monoacetylate spermidine and will diacetylate spermine. Both monoacetyl spermidine and diacetylspermine are substrates for export by DAX in mammalian cells, as depicted in Fig. 5.

Diacetylspermine

Fig. 5. Relationships between metabolism of intracellular amines and export of specific amines by the diamine exporter (DAX). Decarboxylation of lysine (Lys) and ornithine (Orn) by ODC produces the diamines cadaverine (Cad) and putrescine (Put), respectively. Putrescine is the precursor for spermidine (Spd) formation, which can be acetylated at either the N1 (N'AcSpd) or N8 (N8AcSpd) positions. MDL 72.521 is an inhibitor of the FAD-dependent polyamine oxidase (PAO), and verapamil (Vpm) is an inhibitor of DAX. This figure includes the general chemical structure of the amines exported by DAX. (Reprinted with permission from ref. 47.)

Fig. 5. Relationships between metabolism of intracellular amines and export of specific amines by the diamine exporter (DAX). Decarboxylation of lysine (Lys) and ornithine (Orn) by ODC produces the diamines cadaverine (Cad) and putrescine (Put), respectively. Putrescine is the precursor for spermidine (Spd) formation, which can be acetylated at either the N1 (N'AcSpd) or N8 (N8AcSpd) positions. MDL 72.521 is an inhibitor of the FAD-dependent polyamine oxidase (PAO), and verapamil (Vpm) is an inhibitor of DAX. This figure includes the general chemical structure of the amines exported by DAX. (Reprinted with permission from ref. 47.)

Elevated levels of polyamine metabolic genes, including both ODC and SSAT in prostate cancers, will conceivably lead to enhanced export of diacetylspermine. Depending on its fate after export, this amine could be detected in a patient's urine. Studies of polyamine export, which used inverted plasma membrane vesicles, indicated that unmodified polyamines were poor substrates for DAX (47). Thus, unmodified spermidine and spermine would find their way into urine either as a consequence of cell disruption or DAX-independent export mechanisms. Diacetylspermine appears to be the predominant form of urinary polyamines, and levels of this amine appear to increase significantly as a consequence of carcinogenesis. The studies of Kawakita and colleagues suggest that urinary levels of diacetylspermine may be a useful prognostic marker for several epithelial cancers. Whether measurement of urinary diacetylsper-mine can be used as a predictive marker of DFMO effect in cancer chemoprevention remains to be determined. Providing urine samples may be more widely acceptable to the general public, compared with allowing invasive procedures to obtain tissue samples. Thus, the search for surrogates for measures of tissue polyamine pools continues.

13. Summary and Conclusions

Measures of polyamine metabolism include measurements of gene products that regulate polyamine metabolism and sizes of individual polyamine pools. Several genes involved in polyamine metabolism are known targets of pathways regulating normal growth and development and tissue repair and have been implicated in carcinogenesis. However, assays that measure changes in these gene products have not proven to be reliable markers of cancer progression. This shortcoming relates, in part, to substantial variability in the values of these parameters between individuals for any specific tissue. Measures of polyamine pool size are also variable for any given tissue between individuals. However, interindividual variability in measures of polyamine pool size are less than for other polyamine parameters. Measures of changes in polyamine pool sizes are also validated as markers of effect for the cancer chemopreventive agent DFMO. The spermidine:spermine ratio appears to be the most reliable marker for DFMO in studies of colon cancer chemoprevention. Current cancer chemoprevention trials can use measures of polyamine pools to identify the lowest, and thus probably safest, DFMO dose that will confirm biochemical activity of DFMO in a desired target tissue. Measures of polyamine pool sizes may also have relevance to selection of adequate doses of other agents, including some NSAIDS. Future studies should evaluate markers of polyamine metabolism in sources other than target tissues, as these are difficult to obtain. Urinary levels of diacetylated polyamines may prove to be useful as markers of cancer progression and as predictive markers for drugs affecting polyamine metabolism.

Acknowledgments

The authors thank the many physicians and nurses who have contributed to these studies, especially our long-time collaborator Frank L. Meyskens Jr. Other key contributors include Lee Hixson, Harinder Garewal, Westley Lagerberg, Richard Sampliner, David Earnest, Dan Pelot, Steve Goldschmid, Ann Booth, and Peter Lance. The work described in this chapter has been generously supported by the National Institutes of Health.

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