Polyamine levels, ornithine decarboxylase (ODC) activity, and ODC-mRNA expression in normal and cancerous human colonocytes

Life Sciences, vol. Printed in the USA

50, pp. 1417-1424

Pergamon Press

POLYAMINE LEVELS, ORNITHINE DECARBOXYLASE (ODC) ACTIVITY, AND ODC-mRNA EXPRESSION IN NORMALAND CANCEROUS HUMANCOLONOCYTES. Y. Elitsur, I J.A. Moshier, R. Murthy A. Barbish, and G.D. Luk. Department of Medicine Harper-Grace Hospitals and Wayne State University Detroit, Michigan (Received in final form February 25, 1992)

Summary Ornithine decarboxylase (ODC) and polyamines (putrescine, spermidine, and spermine) are crucial for cell proliferation. Recently, elevated ODC activity and polyamine levels have been suggested as biological markers for human colon cancer. In this study, we measured ODC activity and the levels of polyamines (putrescine, spermidine, spermine, and cadaverine) and acetyl-putrescine in human colonocytes isolated from cancerous areas compared to the adjacent normal colon tissue. In addition, ODC mRNA expression was compared between both groups. We found that colonocytes isolated from cancerous areas had significantly higher mean value of ODC activity, putrescine, spermidine, spermine, and cadaverine levels up to 1480%, 4?0%, 260%, 380%, and 510% respectively compared to colonocytes isolated from the adjacent normal colonic mucosa. No difference was found in acetyl-putrescine levels between cancerous and normal colonocytes. Steady-state levels of ODC mRNAwere slightly elevated in cancerous colonocytes relative to normal colonocytes in two of three paired samples. However, the increase in ODCmRNAlevels is not sufficient to account for the increase in ODC activity suggesting that colonocyte ODC activity is regulated post-transcriptionally. Gastrointestinal tract malignancy is s t i l l one of the leading causes of mortality and morbidity in the United States as well as in the western world. Unfortunately, in spite of all therapeutic modalities known for this disease, prognosis is s t i l l poor. The delayed clinical symptoms in this disease leading to late diagnosis is one of the major contributing factors to the given prognosis. Ornithine decarboxylase (ODC) and polyamine biosynthesis are crucial for cell proliferation in the human gut epithelium. Previous data have shown that ODC activity correlates with increased proliferation in human and in animal models (1). Increased levels of ODC activity were found in different hyper-

iDr. Elitsur's current address: Marshall University School of Medicine, Department of Pediatrics, Division of Gastroenterology, Huntington, WV 257010195

Copyright

0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights reserved.

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proliferative states of the gut epithelium such as post-starvation and during rapid development (2,3). ODC was previously suggested as an early marker for cell proliferation and hence may be used as a marker for early detection of human colon cancer. We (4) and others (5) have reported a higher ODC level in the normal mucosa compared to the cancer mucosa of the colon in human and animal models. Moreover, i t has been previously shown that ODC a c t i v i t y correlates with the degree of dysplasia in human colon mucosa (6,7). We have shown that ODC activity correlates with the degree of colonic dysplasia in patients with familial polyposis (8). Concurring with previous reports (6,7), we also found correlation between the ODC level and mucosal dysplasia in patients with colorectal cancer (9). Unfortunately, most of the human studies correlating mucosal ODC level and mucosal dysplasia were done in colonic biopsy specimens. Since ODC is measured per mg of protein in the sample, a significant error may occur when nonepithelial components such as sub-mucosa, lamina propria lymphocytes and other tissues participate in the ODC calculation. This may also explain the wide v a r i a b i l i t y in ODC levels measured in biopsies of human colons. To avoid this p i t f a l l in this study, we investigated the ODC a c t i v i t y and the polyamine level in human colonocytes isolated from normal and cancerous areas of patients who underwent colon resection. Moreover, we have also measured the steady-state levels of ODC mRNA in the colonocytes from three pairs of mucosa. We found that ODC level was higher in cancer colonocytes compared to normal colonocytes. Significant differences were also found in polyamines [putrescine (Put.), spermidine (Spd.), spermine (Spm.), and cadaverine (Cada.)], but not in acetylputrescine (Ac-Put.) levels between normal and cancer colonocytes. Therewas no direct correlation between the ODC activity and ODC mRNA level in the colon mucosa, suggesting that the regulation of ODC activity is mostly post-transcriptional. M a t e r i a l s and Methods

Tissue culture media, antibiotics-antimycotic solution (penicillin 10,000 U/ml, amphotericin-B 25 ug/ml, streptomycin 10 mg/ml), and supplies were obtained from GIBCO, Inc., Grand Island, NY. Dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), and other chemicals were purchased from Sigma Chemical Co., St. Louis, MO. Radioactive L-14C ornithine (50 mCi/mmol) was purchased from NEN DuPont, Boston, MA. Colon specimens s t u d i e d were from a t o t a l o f 13 p a t i e n t s admitted to Harper H o s p i t a l f o r bowel r e s e c t i o n f o r colon cancer. Seven o f the p a t i e n t s were male and s i x were women, and t h e i r ages ranged From 44 to 93 years (mean 70). T h e i r colon cancer d i s t r i b u t i o n was as f o l l o w s : cecum - 5, r i g h t colon - 3, t r a n s v e r s e 1, sigmoid - 2, and rectum - 2. Duke c l a s s i f i c a t i o n d i s t r i b u t i o n was 3 w i t h Duke-A, 5 w i t h Duke-B1, 1 w i t h Duke C-1, 2 w i t h Duke-C2, and 2 w i t h Duke-D. M a c r o s c o p i c a l l y normal and cancerous colon s e c t i o n s from each p a t i e n t were t r a n s f e r r e d in i c e - c o l d RPMI-1640 medium to the l a b o r a t o r y w i t h i n 30 minutes a f t e r s u r g i c a l r e s e c t i o n . Only colon specimens which were s u b s e q u e n t l y confirmed histologically were used in t h i s s t u d y . The study was approved by the institutional Human I n v e s t i g a t i o n Committee. I s o l a t i o n of c o l o n o c y t e s was done according to the method o f B u l l and Bookman (10). B r i e f l y , the mucosa from cancerous and a d j a c e n t normal areas were separated from the musculature l a y e r and were minced i n t o small pieces (0.5 x 0.5 cm). The Fragments were t r e a t e d s e q u e n t i a l l y w i t h calcium-magnesium f r e e Hanks' balanced s a l t s o l u t i o n (CMF-HBSS) c o n t a i n i n g 5 mM DTT f o r 10 min to remove the mucus l a y e r . E p i t h e l i a ] c e l l s were removed by t r e a t i n g the t i s s u e w i t h CMF-HBSS c o n t a i n i n g 0.75 mM EDTA, 5% f e t a l c a l f serum (FCS) and a n t i b i o t i c - a n t i m y c o t i c

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solution (GIBCO, 1% v/v) for 60 min. Culture media was changed every 20 min After each step, the supernatant was collected and then washed (x3) with HBSS. Epithelial cells from all isolated fractions were combined for further analysis. Addition of peptidase inhibitor (phenylmethylsulfonyl fluoride) to the media during the isolation preparation affected the colonocyte separation yield but did not affect v i a b i l i t y , hence i t was not used in the subsequent experiments. The colonocytes were then pelleted and stored in -70°C for ODC and polyamine analyses. Purity and v i a b i l i t y were checked by trypan blue exclusion and light microscopy examination. V i a b i l i t y was found to be greater than 85% in all cell fractions. Isolated colonocytes were homogenized in guanidine monothiocyanate and total RNA was isolated by direct precipitation in the presence of lithium chloride (11). RNA samples were fractionated by electrophoresis in 1.5% agarose gels containing 6% formaldehyde and 20 mM sodium phosphate, pH 6.8 and subsequently transferred to Gene Screen membranes (New England Nuclear, Boston, MA). The relative amounts of 28S and 18S ribosomal RNA in each lane was determined by scanning a photographic print of the ethidium bromide-stained RNA with a Zeineh Video Densitometer (BioMed Instruments, Inc., Fullerton, Ca). HumanODC cDNA probe was nick-translated with [a-32P] dCTP and hybridized to f i l t e r s in SSPE (750 mM NaCl, 50 mM sodium phosphate, 5.0 mM EDTA adjusted to pH 7.4 with 10 M NaOH), 50% formamide, 5x Denhardt's solution, and 10% dextran overnight. Filters were washed with lx SCC (15 mM sodium citrate, 150 mM NaCl, pH 7.0) at 60°C and exposed to XOMATfilm for five days. Relative ODCmRNAlevels were estimated by densitometric scanning of the resulting autoradiograph. The intensities of the radioactive signals hybridizing to ODC mRNA were normalized to ribosomal RNA levels. ODC activity was determined using the method of Beaven et al. (12), as described (13,14). Briefly, normal and cancerous colonocytes were collected by centrifugation and the pellets were homogenized in a buffer containing 50 mMTris (pH 7.5), 250 mM sucrose, 0.1 mM EDTA, 0.4 mM pyridoxal 5' phosphate, and 1 mM DTT. Homogenateswere centrifuged at lO0,O00g for 60 minutes and the supernatants were assayed for ODC activity. The incubation mixture contained 50 mM HEPES, 1 mM EDTA, 0.25 mMpyridoxal 5'phosphate, 1 mMDTT, 130 uM ornithine, and 0.144 uM 14C-ornithine. Bradford's protein-dye method was used to determine protein concentration in the assay mixture (15). Polyamine levels were measured in the normal and cancerous pellet homogenates according to the method of Kabra et al. (16). Briefly, perchloric acid (0.6 N) was added for at lease 30 min at 4°C to precipitate proteins. The supernatant was collected and pH was adjusted between 9 and 11 with saturated sodium carbonate. The supernatant with dansyl chloride and internal standard, 1,6 diaminohexane, was incubated at 70°C for 10 min. After incubation, excess dansyl chloride was removed by derivatization with L-proline. Impurities were removed by u t i l i z i n g the Bond-Elute C18 precolumn and then separated on a reverse phase, C18, 3u column with gradient elution. Detection limits range between 200 and 300 fmole for unconjugated and acetylated polyamines. Polyamine quantitation was calculated using peak areas and the internal standard. Due to the wide v a r i a b i l i t y among tissue samples, a non-parametric s t a t i s t i c analysis (Wilcoxon test) (17) was used to evaluate the difference between the cancerous and normal colonocytes. "P" values below 0.05 were considered significant.

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Results ODC a c t i v i t i e s were s i g n i f i c a n t l y higher in the human colonocytes isolated from the cancer area compared to colonocytes isolated from a normal area in the same patients (p
4

ODC activity in human colonocytes

• •

01

.i i

~

0

(Q 0

2

0

]

I

Normal

Cancer

Fig. 1 Fig. 1: ODC a c t i v i t y in human colonocytes. Human colonocytes were isolated from cancer and adjacent normal colonic mucosa as described in Materials and Methods. ODC a c t i v i t y (pmol COJmg/hour protein) was 1.6 to 47.2 (mean 14.8) fold higher in cancerous colonocytes compared to normal colonocytes. Results represents the log. mean ± SEM of 13 pairs of specimens (normal and cancer), each done in t r i p l i c a t e determinations. *pO.05). Total RNA was isolated from three paired samples of human cancerous and normal colonocytes and probed with 32P-labelled ODC cDNA. the i n t e n s i t i e s of the radioactive bands in Figure 3 (bottom) were normalized to the r e l a t i v e amounts of 28s and 18s ribosomal RNA (top) in each sample. Steady-state levels of ODC mRNAwere s l i g h t l y elevated in two cancerous colonocyte specimens r e l a t i v e to the

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Polyamines and ODe in Human Colonocytes

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Polyamine levels in human colonocytes 1200

1000

•

Normal

[]

Cancer

800 600 400

200 Ac-put,

Put.

Spd.

Spm.

Cada.

Polyamines Fig. 2 Fig. 2: Polyamines [putrescine (Put.), spermidine (Spd.), spermine (Spm.), and cadaverine (Cada)] level in human colonocytes. Polyamines determination was done by HPLCanalysis as described in Materials and Methods. The mean putrescine, spermidine, spermine, and cadaverine l e v e l s were higher in human cancerous (372, 218, 465, and 210 nmol/mg protein r e s p e c t i v e l y ) compared to normal colonocytes (120, 160, 291, and 61 nmol/mg protein r e s p e c t i v e l y ) . Results represents a mean ± SEM of at least 9 pairs of specimens, each done in t r i p l i c a t e determinations. *Represents p
NL

CA

NL

CA

NL

ODC (pmol/hr/mg protein

322

67

402

10

359

85

ODC mRNA (Densitometer)

3.6

2.7

6.5

3.3

5.3

5.4

Discussion ODCactivity and polyamine biosynthesis are essential for cell proliferation in many tissues, including the gastrointestinal epithelium. ODC activity was previously found to correlate with tumorigenesis in human and animal models (1,18). Moreover, difluoromethylornithine (DFMO), the specific inhibitor of ODC,

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Polyamines and ODC in Human Colonocytes

CA

NL

CA

NL

CA

Vol. 50, No. 19, 1992

NL

28S

18S

ODC mRNA

1

2

3

4

5

6

Fig. 3 Fig. 3: Human ODC m-RNA l e v e l s in human cancerous and normal colonocytes. Total RNA was i s o l a t e d from 3 pairs of samples and were probed with P-32 r a d i o l a b e l l e d human ODC cDNA. CA - cancerous; NL - normal

has been found to a l t e r p r o l i f e r a t i o n and tumor formation in several experimental models (19,20). In s p i t e of the close association between ODC a c t i v i t y and tumorigenesis, a s i g n i f i c a n t overlap in 00C values between normal and cancerous mucosa e x i s t s , hence decreasing the s p e c i f i c i t y of 00C a c t i v i t y as a marker f o r tumor progression. In t h i s study, we attempted to approach t h i s problem in two ways: (a) To avoid the proteins of n o n - e p i t h e l i a l o r i g i n by using i s o l a t e d human colonocytes, and (b) to i n v e s t i g a t e whether ODC a c t i v i t y c o r r e l a t e s with ODC-mRNA expression. Concurring with previous studies done in human colon biopsies, we also found higher ODC a c t i v i t y i s o l a t e d cancerous colonocytes compared to normal colonocytes. Unfortunately, in s p i t e of the d i f f e r e n t methodology used in our study, a wide v a r i a b i l i t y was found between ODC a c t i v i t i e s in both cancerous and normal

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colonocytes, suggesting that non-specific proteins were not the reason for the wide v a r i a b i l i t y seen in previous reports. We found that ODC mRNA steady-state levels, although slightly elevated in two of three paired samples, did not correlate with ODC a c t i v i t i e s in cancerous and normal colonocytes. Differences in ODC activity up to 40 times were accompanied by a mere two-fold increase in ODC mRNAlevel (Fig. 3 and Table I). These findings are in general agreement with those of Radford et al. (21), who examined the level of ODC mRNAexpression in 18 human colorectal carcinomas and six adenomatous polyps. In all cases, ODC mRNAlevels were greater in tumors than in matched samples of adjacent, pathologically normal mucosa. On the average, increases in ODC mRNAin polyps and carcinomas relative to controls were three- and four-fold, respectively. Again, a direct correlation between ODCmRNA levels and enzyme activities was not observed, but Radford et al. (21) suggested that overexpression of the ODC gene may be important to the maintenance of the neoplastic state. We propose that the quantitative differences between this study and that of Radford et al. are most l i k e l y due to methodological differences in post-surgery tissue manipulation. Furthermore, post-transcriptional regulation of ODC activity has been demonstrated in a number of epithelial cell types (22). I t is thus postulated that ODC activity may also be regulated post-transcriptionally. Whether this is the reason for the low specificity of the ODC activity in human colorectal disease is s t i l l to be determined. Putrescine, spermidine and spermine, are found in all cells of higher eukaryotes (9). Polyamines have been linked to the regulation of DNA, RNA and to the synthesis of protein (23). Polyamines are increased in different high proliferative conditions such as during development (24), post-lactation, and/or after a refeeding period (25). Polyamines were also found to be elevated in pathological hyperproliferative states such as familial polyposis syndrome of tumorigenesis (4,6). Previous data have documented the close r e l a t i o n s h i p between tumor formation and the polyamine biosynthesis pathway. I n h i b i t i o n of various enzymes along the polyamine biosynthesis pathway, i . e . ODC, or S-adenosylmethionine decarboxylase (SAMDC), reduces tumor formation in animal models (26,27). Previous reports on human colon cancer have shown a c o r r e l a t i o n of high ODC a c t i v i t y and polyamine level in human colonic mucosa (7,8,28). Others found elevated levels of Nacetylspermidine in human colorectal mucosa compared to normal mucosa (29). Nonetheless, no c o r r e l a t i o n was found with tumor size, Duke's stage, or h i s t o l o g i c a l grade of the tumor (16). Concurring with those studies, we also found that polyamines are elevated in cancerous colonocytes compared to normal colonocytes. No c o r r e l a t i o n was found between the polyamine levels and the h i s t o l o g i c a l invasion of the tumor (Duke c l a s s i f i c a t i o n ) . In summary, we have demonstrated that human colorectal cancer is associated with increased ODC a c t i v i t y and polyamine l e v e l . Moreover, we demonstrated that increases in ODC a c t i v i t y are not accompanied by p a r a l l e l increases in ODC mRNA steady-state l e v e l . We postulate that ODC a c t i v i t y and polyamine l e v e l s , but not steady-state ODC mRNA l e v e l s , are consistent with a h y p e r p r o l i f e r a t i v e state in isolated colonocytes, and thus, may be used as a marker f o r increase c e l l proliferation. Acknowledgements We acknowledge the assistance of the Pathology Department of Harper Hospital, D e t r o i t MI for t h e i r help in obtaining the human i n t e s t i n a l specimens. We also acknowledge Mrs. Jennifer Long for her e x c e l l e n t s e c r e t a r i a l assistance.

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This work was supported in part by intramural funding from the C h i l d r e n ' s Hospital of Michigan and from Harper Hospital and the Hudson-Webber Foundation. Dr. E l i t s u r is a r e c i p i e n t of the C h i l d r e n ' s Hospital of Michigan Intramural Funding Award. References 1. 2. 3. 4. 5. 6. 7. 8. g.

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I0. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

I

I

21. 22. 23. 24. 25. 26.

27. 28. 29.