Effects of urinary transferrin and ornithine decarboxylase-inducing fraction on rat bladder carcinogenesis

65

Cancer Letters, 45 (1989) 65-70 Elsevier Scientific Publishers Ireland Ltd.

Effects of urinary transferrin and ornithine inducing fraction on rat bladder carcinogenesis S. Noguchi, Department (U.S.A.) (Received (Accepted

Y. Yura,

of Pathology,

15 December 20 December

0. Hayashi,

Northwestern

S. Samma

University

decarboxylase-

and R. Oyasu

Medical School,

303 E. Chicago

Avenue, Chicago,

Illinois 60611

1988) 1988)

Keywords:

Summary

urinary transferrin;

rat bladder

cancer. Using heterotopically transplanted rat urinary bladder (HTB) system, we previously have shown that contact with urine enhanced bladder carcinogenesis initiated by carcinogen. In order to screen urine for promoter substances, several short term in oitro assays were developed and their results were correlated with the in vivo assay results. Chromatographically separated urine fractions were examined for the inability to induce ornithine decarboxylase (ODC), to enhance incorporation of PH]thymidine in a bladder carcinoma cell line (804G) and to form colonies in soft agar by NRK-49F. Data from the ODC assay and soft agar colony formation correlated well with the results derioed from chronic animal studies. Thus then two assays appear useful in further screening urine for promoter substance. Data furthermore indicate that ODC-inducing urine component(s) may play a primary role in the steps following initiation whereas transferrin, a mitogenic urine component, may play a secondary role.

Correspondence

too:R. Oyasu

Introduction

Using the HTB system [ 11, we earlier demonstrated that contact with urine enhanced bladder carcinogenesis as compared with the contact with equiosmolar NaCl solution. The tumor-enhancing activity was subsequently ascribed to crude chromatographic components designated as Fraction I (Fr. I) and Fraction II (Fr. II) [Z]. These fractions were also capable of inducing omithine decarboxylase (ODC) activity in a tester bladder carcinoma cell line (804G) in vitro [2]. Since the promoter activity of Fr. I was much higher than Fr. II, we focused our interest on Fr. I. Further purification of Fr. I using CM-Sephadex chromatography succeeded in separating a component, identified as transferrin (TF), which stimulated DNA synthesis but not ODC induction in 804G 131. The TF-rich subfraction of Fr. I was designated as urinary TF-rich fraction. The present study had two objectives, first to determine how useful short-term in vitro assays may be in predicting the results of longterm chronic in vivo studies in our search for urinary tumor promoting factors, and second, to determine if TF, a mitogenic agent in the in vitro assay, acts as a promoter of bladder carcinogenesis

0304~3835/89/$03.50 0 1989 Elsevier Scientific Publishers Ireland Ltd Published and Printed in Ireland

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66

Hatertals and methods

1. In vitro biological activities of urine samples The samples tested were whole urine, the crude urine fractions (Fr. I and Fr. III) (see Results), urinary TF-rich fraction and authentic rat TF (Cooper Biomedical, Malvern, PA). As a control, 2.1% NaCl was used (Table 1). The amount of test samples added to groups 3,4 and 5 were adjusted by their TF content to receive an amount of TF (30 pg/ml) equivalent to that contained in 2.5 ml portion of whole urine. Fr. III was delivered in the same amount as that of Fr. I (3.0 mg/ml). For group 2, 0.5 ml of whole urine after its osmolality was adjusted to 700 mOsmol/kg was used. A larger amount was not used because of its known toxicity. All test samples had their pH adjusted to 7.0 with 1.0 N NaOH solution. Cell lines

Two cell lines were used in these studies: (1) 804G is a squamous cell carcinoma cell line of urinary bladder from a male AC1 rat and induced in our laboratory [4] by the in vitro technique described by Hashimoto and Kitagawa [5]; (2) NRK-49F purchased from American Type Culture Collection, Rockville , MD. 804G was maintained in MEM with 10% fetal calf serum (FCS), and NRK-49F in DME with 10% calf serum (CS) at 37OC in a humidified atmosphere of 5% CO, and 95% air. Chromatographic separation of rat urine

At 24 h normal rat urine was collected and separated by Bio-GeI P-100 chromatography as previously described [3,4]. The eluate of every third tube was tested for ODC inducibility in 804G and soft agar colony formation by NRK-49F. To obtain the urinary TF-rich fraction, tubes 16-24 of Fr. I of the Bio-Gel-P100 column were combined, lyophilyzed, dissolved in 0.1 M acetate buffer, pH 5.5, and applied to a 1.6 x 25 cm CM-Sephadex C-50 (Pharmacia, Inc., Piicataway, NJ) column equilibrated with the buffer. After elution of unabsorbed fractions with the buffer, absorbed fractions were eluted with a linear gradient 0 to

1.0 M NaCl in the buffer. Absorbed fractions which stumulated i3H]thymidine incorporation but did not induce ODC of 804G were pooled, dialyzed against distilled water and lyophilyzed. This urine sample was used as a urinary TF-rich fraction. PH]Thymidine incorporation in 804G cells 804G cells were plated in 24-well plates

(Becton-Dickinson, Oxnard, CA) in MEM with 10% FCS at a density of 1 x lo4 cells/ml/ well. Three days later, the medium was changed to MEM with 0.2% FCS after washing twice with Hanks’ balanced salt solution (HBSS). Fifty microliters of test samples were added to each well. After an 18 h incubation, cells in triplicate were pulsed for the next 24 h with 0.1 &i ~3H]thymidine (Amersham Corp., Arlington Heights, IL) and the radioactivity incorporated into trichloroacetic acid-precipitable material was determined by a scintillation counter (Beckman Instruments, Irvine, CA) [3].

ODC assay 804G cells were plated in 60-mm dishes at a density of 1 x lo5 cells in MEM with 10% FCS. After 4 days the medium was changed and the 5-day-old cultures were used for ODC assay. Cells were washed once with MEM and incubated with a mixture composed of 100 ~1 of test sample and 5 ml of MEM containing 10 mM HEPES. After 5 h of incubation, cells were washed with ice-cold PBS, pH 7.4, 3 times, frozen on solid CO, ethanol, thawed and harvested into centrifuge tubes by scraping with rubber policeman. Cells from two dishes were combined and disrupted by two cycles of freezing, thawing and sonication. After centrifugation at 20,000 x g for 20 min at 4OC, the supernatant was harvested and the ODC activity was measured by the method described previously [4].

Soft agar

assay

A modified soft agar colony assay [6] prepared with a basal layer of 0.4 ml of supplemented DME medium in 0.6% agar fortified

67

with 10% CS placed in 24-well plastic dishes. The second layer consisted of 0.5 ml of supplemented DME medium in 0.3% agar fortified with 10% CS and 5 X lo3 NRK-49F cells and 25 ~1 of test sample. The third layer was composed of 0.1 ml of the 0.3% agar-DME without test sample. After 2 weeks of culture at 37OC in a humidified atmosphere of 5% CO, and 95% air, colonies consisting of more than 8 cells were counted. Data represent the average. All in vitro assays were repeated at least once. 2. In vivo assay using HTB system A total of 286 male Fischer 344 rats weighing 130-150 g (Harlan Sprague-Dawley Inc., Indianapolis, IN) were housed 4 to 5 per cage in an air-conditioned room at 22OC with 50% humidity and a 12 h light-dark cycle. They had free access to tap water and commercial stock diet (Purina 5012, Ralston Purina Co., St. Louis, MO). One-half of the animals served as donors of urinary bladders. They were transplanted into the gluteal muscle of recipient animals by the technique described previously [l]. The experimental design is shown in Fig. 1. Following a single dose (0.25 mg) of ~-methyl-~-ni~osourea (MNU, ICN Pharmaceuticals, Plainview, NY) administered -4

0 1

-cr

12

2OWKS

x

x

1

Fig. 1. Experimental design of in vivo study. Four weeks after transplantation of bladder, rats received into the I-fTBs a single dose of MNU followed 1 week later by weekly instillation of test samples. V MNU 0.25 mg, q 0.9% NaCI, X sacrifice, Q test samples: Group 1,2.1% NaCl (control); Group 2, whole urine; Group 3, TF (6 ccg/ml); Group 4, TF (30 pg/ml); Group 5, urinary TFrich fraction (15 pg/ml); Group 6, urinary TF-rich fraction (75 pg/ml); Group 7, Fraction III (0.6 pg/ml); Group 8, Fraction III (3.0 rg/ml); Group 9, Fraction I (0.6 pg/ml); Group 10, Fraction I (3.0 pg/ml).

into the HTB through an attached reservoir, rats were divided into 10 groups. Each rat received into the HTB various test urine samples once a week for up to 20 weeks (Fig. 1). Group 1 received 0.5 ml of 2.1% NaCl solution (equiosmolar to urine used) once a week. Group 2 received 0.5 ml of rat urine whose osmolality was adjusted to 700 mOsmol/kg and pH to 7.0. Test material administered to groups 3,5,7, and 9 was equivalent in amount to that contained in 0.5 ml of urine, whereas 5 times as much material was tested in groups 4,6,8, and 10. These two concentrations were chosen to ensure their effects. Groups 3 and 4 received chromatographically purified rat TF. The amount of urinary TF-rich fraction administered to group 5 (15 pglml) and the amount of Fr. I for group 9 (0.6 mglml) were both based on their TF content, and was equal to that in group 3. To groups 7 and 8 were added Fr. III (see Results) and received the amount equivalent to those used for the groups receiving Fr. I. All test samples were dissolved in 2.1% NaCl solution before use. One-half of the animals, chosen at random, were killed after 12 weeks and the remaining rats after 20 weeks of weekly treatment. After 24-h fixation in 10% neutral buffered formalin, the bladders were examined for gross alteration, cut into 5 to 7 longitudinal pieces and submitted for microscopic examination. Bladder lesions were evaluated by the criteria previously described [ 11. Results

1. Separation of Fr. 1 and Fr. Ill Lyophilized crude urine was applied onto a Bio-Gel P-100 column. The aliquots of the fractions were assayed for ODC inducibility in 804G cells. Two ODC inducing peaks previously designated as Fr . I and Fr. II were identified in the high and low molecular weight regions, respectively [Fig. 21. When these fractions were tested for the soft agar colony formation by NRK-49F, an excellent correlation between ODC inducibility and colony formation was found [Fig. 21.

Fr.lJI

I

Fraction

Number

Bio-Gel P-100 chromatography of rat urine. Three-hundred milligrams of dialized and subsequently lyophilized Fig. 2. urine sample dissolved in 4.0 ml of 0.1 M Tris-HCI buffer, pH 7.5, with 10% ethylene glycol were applied to a 2.6 x 100 cm Bio-Gel P-100 column equilibrated with the buffer. Each eluate was collected in 4.5 ml portions, monitored by absorbance 280 nm ( 0 0). Fr. I (tube No. 16-24), Fr. II (tube No. 37-54), Fr. III (tube No. 25-36). Each fraction was tested in duplicate for ODC inducibility in 804G cells (AA) and soft agar colony formation by NRK-49F cells (0- - - - -0) as described in “Materials and methods”.

In vitro biological activities of urine samples. Table 1. Samples were done in triplicate and the data represented

All three assays were done by the methods described the average number and standard deviation.

in the text.

Samples

[3H]TdR incorporation (CPM/culture)

ODC inducibility (CO, nmol/mg protein/min)

Soft agar colony formation (colonies/well)

1

12,228

+ 360

0.48

f 0.04

0

4,364b 49,353b

f 322 + 4763

0.90 0.34

-+ 0.41 f 0.04

154b 0

44,204b

+ 9973

0.57

-c 0.12

0

2 3 4 5 6

2.1% NaCl (control) Whole urine TF (30 &ml) UTF Fraction” (75 i&ml) Fraction I (3.0 mg/ml) Fraction III (3.0 mg/ml)

+ 21

25,953b,d f 2704

3.43b.e * 0.21

212b.’ * 7

14,875

1.27b

lllb

+ 976

f 0.07

“UTF fraction, urinary transferrin-rich fraction, bP < 0.001, and ‘P < 0.01 by Student’s < 0.05, and 9 < 0.01 compared with Fraction III (Student’s t-test).

f-test compared

f 8

with control, dP

69

Table2 Group no. and treatment

Microscopic findings of HTBs following weekly instillation of test samples. HTB findings (12 weeks) No. of rats

1 2.1%NaCl 8 (control) 2 Whole urine 3 TP (6 pg/ml) 4 TF (30 Ccs/ml) 5 UTFfractionb (15 pg/mI) 8 6 UTP fraction (75 rs/ml) 7 Fraction I 8 (0.6 m&ml) 8 Fraction I 8 (3.0 mglml) 9 Fraction III 7 (0.6 mgfml) 10 Fraction III 8 (3.0 mg/ml)

HTB findings (20 weeks)

Normal

SHI,

NPH,

CA

No. of rats

Normal

SHa,

NPH,

CA

8

0

0

0

5

4

1

0

0

1 0 0 0

0 0

5 6 7 7

0

1 0

1 0 0 0

4 6 3

4 2 1 3

1 0 0 1

2 0 0 0

8

0

0

0

7

6

1

0

0

7

0

0

1

8

2

4

0

1

1

7’

2

3

7

1

4

1

5d.e

6

1

0

0

7

6

0

1

1

7

0

1

1

7

6

1

1

0

*SH: simple hyperplasia, NPH: nodulopapillary hyperplasia, and CA: carcinoma (ail carcinomas arised were grade I, stage 0, transitional cell carcinoma), bSee Table 1 for definition, T = 0.001 as compared to Groups 1 and 10 (Fisher’s exact test), dP = 0,021 as compared to Group 1 (Fisher’s exact test), ‘P = 0.01 as compared to Group 10 (Fisher’s exact test).

The broad region (tube numbers 24-36) next to Fr. I was designated as Fr. III. It induced ODC activity and stimulated colony formation less efficiently than Fr. I. The TF content of Fr. III was less than 0.002 pg/‘ml as measured by enzyme linked immunosorbent assay [7f. 2. In vitro biological activities of urine samples

Authentic TF and the urinary TF-rich fraction significantly stimulated thymidine incorporation (P < O.OOl), but not ODC inducibility or soft agar colony formation (Table 1). Fr. I showed significantly higher values than Fr. III in all three assays. Whole urine stimulated colony formation but inhibited thymidine incorporation.

3. Effects of urine samples on rat urinary bladder curcjnogenes~s

Animals receiving the high concentration of fr. I (group 8) showed a significantly higher incidence of simple hyperplasia at 12 weeks and carcinoma at 20 weeks than those receiving Fr. III (group 10) (P = 0.001and 0.01, respectively) or saline (group 1) (P = 0.001 and 0.021, respectively) (Table 2). Authentic rat TF and urinary TF-rich fraction did not enhance the MNU-initiated tumorigenesis. Discussion The goal of the present investigation was to determine whether short-term in vitro assays (ODC induction, [3H]TdR incorporation and

70

soft agar colony formation) are useful in predicting the long-term in vivo effects of rat urine components, because availability of such tests is desirable before a long-term animal study is initiated. Our earlier studies showed that ODC induction in target cells was an important property shared by crude urine fractions which were capable of enhancing carcinogen-initiated rat bladder carcinogenesis. From one of these two crude fractions, TF was isolated. TF was found to be a potent growth stimulator of bladder carcinoma cells in culture but to be unable to stimulate ODC. Since cell proliferation is an important property of promoter substances, it is logical to test TF for a promoter role in urinary bladder carcinogenesis in vivo. As an armamentarium for the assays to screen urine for tumor promoters, soft agar colony formation assay was added, because normal urine is known to contain growth factors which can stimulate colony formation of NRK-49F E91. Results show that Fr. I was the most active in all three in vitro assays and in enhancing MNU-initiated tumorigenesis, confirming our previous observation [Z] , whereas Fr. III whose ability to induce ODC and form colony was significantly less than that of Fr. I was only marginally effective in enhancing bladder tumorigenesis. In contrast, authentic TF and urinary TF-rich fraction which were mitogenic but not able to induce ODC or colonies in soft agar, did not enhance bladder tumorigenesis. This may not be surprising: unlike other growth factors, the function of TF is to deliver iron atoms to the cells which are actively proliferating and therefore are in need of iron [lo]. Carcinogen-initiated cells in the HTB may not need additional supply of iron until they are stimulated to grow. Growth simulation may be provided by the growth factor(s) which enhance ODC inducibility of S04G cells and soft agar colony formation of NRK-49F cells. Thus the ODC induction and the soft agar colony formation assays appear useful in further screening urine for promoter substance(s). Our preliminary data suggest that epidermal growth

factor and its related growth factor may be responsible for ODC induction. Further study is in progress to determine whether the promoting activity of Fr. I is due to ODC-inducing substance(s) alone or it requires in addition, urinary-rich TF fraction. Acknowledgment The investigation was supported by NIH grant CA 14649. We acknowledge Mary Kelly for her expert technical assistance. References 1

2

3

4

5

6

7

8

9

10

Oyasu, R., Iwasaki, T.. Matsumoto, M., Hirao, Y. and Tabuchi, Y. (1978) Induction of tumors in heterotopic bladder by topical application of N-methyl-N-nitrosourea and N-butyl-N(3-carboxypropyl)nitrosamine, Cancer Res., 38,3019-3025. Babaya, K., Lrumi, K., Ozone, S., Miyata, Y., Mortkawa, A., Chmiel, J.S. and Oyasu, R. (1983) Capability of urinary components to enhance ornithine decarboxylase activity and promote urothelial tumorigenicity, Cancer Res., 43, 1774-1782. Hayashi, O., Noguchi, S. and Oyasu, R. (1987) Transferrin as a growth factor for rat bladder carcinoma cells in culture, Cancer Res., 47,4560-4564. Izumi, K., Hirao, Y., Hopp, L. and Oyasu, R. (1981) In vitro induction of ornithine decarboxylase in urinary bladder carcinoma cells. Cancer Res., 41,405-409. Hashimoto, Y. and Kitagawa, H.S. (1974) In vitro neoplastic transformation of epithelial cells of rat urinary bladder by nitrosamines. Nature (Land.), 252,497-499. Morita, H., Noda, K., Umeda, M. and Ono, T. (1984) Activities of transforming growth factors on cell lines and their modification by other growth factors, Gann, 75, 403 -409. Voller, A., Bidwell, D.E. and Bartlett, A. (1976) Enzyme immunoassays in diagnostic medicine. Bull. WHO, 53, 55 -65. Twardzik, D.R., Sherwin, S.A., Ranchalis, J. and Todaro, G.J. (1982) Transforming growth factors in the urine of normal, pregnant, and tumor-bearing humans. J. Natl. Cancer Inst., 69, 793-798. Stromberg, K. and Hudgins, W.R. (1986) Urinary transforming growth factors in neoplasia: Separation of rz51labeled transforming growth factor-u from epidermal growth factor in human urine. Cancer Res., 46, 60046010. Aisen, P. and Listowsky, I. (1980) Iron transport and storage proteins. Annu. Rev. Biochem., 49.357-393.