Induction of Hyperplasia and Its Suppression by - Cancer Research

[CANCER RESEARCH 37, 1421-1427, May 1977]

SUMMARY

Urinary bladders from Fischer rats organ cultured in achemically defined medium, Ham's F12, underwent transitional cell hyperplasia which persisted for the duration ofthe culture period (10 days). The hyperplastic response wasinitiated at 2 days of culture in basal epithelial cells asevidenced by [3H]thymidine autoradiography. After 2 days,cells classified as intermediate cells were observed replicating DNA in increasing numbers, whereas the frequency ofbasal cells replicating DNA decreased. The peak periods ofbasal and intermediate cell DNA replication were at 2 and 5days, respectively. The total increase in the number of cellsin the epithelium during a 10-day culture period was approximately 2.6-fold. The appearance of DNA-replicatingcells before the appearance of mitotic figures indicated thatthe cells of the transitional epithelium are primarily G1cells.

The hyperplastic response in the transitional epitheliumwas significantly inhibited by hydrocortisone. Epithelia cultured in the presence of hydrocortisone also displayed lessatypia than those epithelia cultured in its absence. Hydrocortisone concentrations of 2.1 and 21 @Minhibited hyperplasia by 75 and 84%, respectively. Cells replicating DNA at2 days of culture were considerably less sensitive to thehydrocortisone inhibition of [3H]thymidine incorporationinto DNA than cells replicating DNA at 4 days of culture. Thepossibility is discussed that basal and intermediate cellsmay have different sensitivities to hydrocortisone.

INTRODUCTION

There have been numerous reports in the literature whichdemonstrate the existence of multiple noninvasive microscopic lesions, ranging from hyperplasia through increasing degrees of epithelial atypia to carcinoma in situ, in areasof cystoscopically normal-appearing human bladder epithehum which lie peripheral to invasive tumors (1, 4, 9, 13, 18—21). The implication of these observations is that some ofthese lesions may be precursors to invasive cancer and thatthey exist for some period of time before its appearance.Recent evidence (5) demonstrates more definitively thathuman bladder cancer develops over a considerable periodof time and is preceded by a preneoplastic phase characterized by progressive degrees of epithelial hyperplasia, dysplasia, and nuclear atypia. A knowledge of the develop

Received October 5, 1976; accepted February 10, 1977.

mental process by which these conditions are established inthe transitional epithelium would add considerably to ourunderstanding ofthe preneoplastic state. Unfortunately, theanalysis of this problem in man is difficult because of theapparently long duration and gradual development of preneoplasia. The problem in man has therefore remainedpoorly understood; this is also the case for experimentalcarcinogen-induced preneoplasia of the bladder. An expenimental system, in which the preneoplastic phase of cancerdevelopment is accelerated under carefully controlled conditions, would be of considerable help in studying thisclinical problem.

In an earlier paper (16), procedures were described forthe successful organ culture of normal and carcinogentreated rat urinary bladder in various chemically definedculture media. One medium, Waymouth's MB752/1 (25),allowed the long-term culture of transitional epithelium. Asmall degree of basal cell hyperplasia was observed ; however, the epithelium remained transitional. This was not thecase in Ham's Medium F12 (8). Although long-term cultureswere maintained, the epithelium displayed moderate tomarked hyperplasia with dysplasia and produced extensivenodular downgrowths into the submucosa.

Although the etiology of hyperplasia and dysplasia in thepreneoplastic condition is obviously quite different fromthat observed in Medium F12, the developmental mechanisms by which hyperplastic and dysplastic states are established in the 2 conditions may be quite similar. A study ofthe response of bladder epithelium to growth in MediumF12 would thus provide useful information for understand

ing the development of preneoplastic lesions in the transitional epithelium. More importantly, the system provides ameans of testing agents which may be potentially useful inthe control or reversal of preneoplastic bladder lesions. Astudy of the response of bladder epithelium in Medium F12was therefore undertaken to determine how hyperplasiaand dysplasia develop and how they can be controlled. Inthis paper, an analysis of hyperplasia and its control byhydrocortisone is presented.

MATERIALS AND METHODS

Animals. Male Fischer 344 rats were obtained from theCharles River Breeding Laboratory, Wilmington, Mass., andused at 7 weeks of age.

Organ Culture Procedure. Animals were anesthetizedwith ether. Urinary bladders were removed aseptically and

1421MAY 1977

Induction of Hyperplasia and Its Suppression by Hydrocortisonein Organ-cultured Rat Urinary Bladder

David H. Reese, Rosalind D. Friedman, and Michael B. Sporn

Lung Cancer Branch, National Cancer Institute, NIH, Bethesda, Maryland 20014

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D. H. Reese et al.

cut into equal halves, longitudinally. These hemibladderexplants were cultured, with the epithelium positioned donsally, on stainless steel grids in 60-mm plastic culture dishescontaining 3 ml of Ham's Medium F12 (8). The mediumcontained 100 units of penicillin and 100 j@gof streptomycinpen ml. Cultures were maintained at 34.5°in a gas phase of4.5% CO2in air. Medium was changed every 2 or 3 days.

In experiments investigating the effects of hydnocortisoneon hyperplasia, sterile aqueous hydnocortisone-21 -sodiumsuccinate (The Upjohn Co., Kalamazoo, Mich.) was used. Ateach change of medium, the hormone was added as afreshly prepared solution. Insulin (crystalline, bovine; EliLilly and Co., Indianapolis, md.), prepared as a sterile stocksolution in 4 mM HCI, was included at a concentration of 1@tg/mlin all media used in hydrocortisone experiments,

including control media.(3H]TdR' Labeling of DNA. Tissues were labeled with 15

pCi of [3H]TdR (specific activity, 20 Ci/mmole; New EnglandNuclear, Boston, Mass.) in 3 ml of medium. Tissues to beused for autonadiography were labeled for 1 hr. and tissuesfor specific activity measurements were labeled for 3 hr.During the labeling periods, dishes were tilted to ensurethat the tissues were completely submerged in the labelingmedium.

Histology and Autoradiography. Tissues were fixed inbuffered neutral formalin, embedded in glycol methacrylate, and sectioned at 2 @m.For autoradiography, sectionswere coated with Kodak NTB2 liquid emulsion and storedfor 14 days at 4°.Autonadiognaphs were developed for 5 mmin D19 Kodak developer, rinsed in water, and fixed for 10mm in Kodak fixer. Sections were stained with hematoxylmnand phloxine B.

Scoring of Data. To quantitate the hyperplastic response,the numbers of nuclei, labeled nuclei, and mitotic figuresper 150-gm length (unit length) of epithelium were scoredon 1 section from each of 4 or 5 explants/time point. Onlyflat unit lengths of epithelia were counted. Folded andinvaginated areas of epithelia were not counted. In the caseof nodular downgrowths which were frequently encountered at 7 and 10 days of culture, if part of a unit length wasflat but also contained a nodular area, it was counted. If,however, the unit length encompassed a length of epithehum which was entirely nodular, it was not counted.

In the area of the cut surface, a small portion of thebladder epithelium becomes attenuated and displays anabnormally high labeling and mitotic index. These woundareas were not scored. A nucleus was considered labeled if7 on more exposed grains were observed in the emulsionabove it. In cases where the grain density above a nucleuswas such that the grains coalesced but with obviously numerous grains present, the nucleus was scored as labeled.Labeled nuclei and mitotic figures were expressed as pencentage of total cells, while nuclear number was expressedas number per unit length.

The distinction between basal and intermediate cells wasmade according to their position in the epithelium. Basalcells were those cells most proximal to the epithelial-sub

I The abbreviation used is: [3H]TdR, [3H]thymidine.

mucosal border. All other cells, which were separated fromthe bonder by another cell (basal cell), were classified asintermediate cells.

Specific Activity of [3H]TdR Incorporation into DNA. Afterlabeling, tissues were held on ice in 3 ml of Dulbecco'sphosphate-buffered saline (3). Each tissue was transferredto a plastic dish containing 0.5 ml of cold calcium- andmagnesium-free phosphate-buffered saline containing 20mM EDTA, and the epithelium was scraped from its collagenous supporting layer with a scalpel. Essentially all of theepithelium was removed by this method without damagingon including submucosal tissues (Fig. 1).

The surface of the dish was washed twice with freshphosphate-buffered saline containing 20 mM EDTA to ensure complete recovery. A total of 1.5 ml of cell suspensionwas obtained . This was made 0.2 N in HCIO4and held on icefor 15 mm. The precipitated cell suspension was centnifuged and washed twice with cold 0.2 N HCIO4.

To the final pellet, 0.6 ml of 1.6 N HCIO4was added, andthe DNA was hydrolyzed for 20 mm at 70°.After centnifugation , the deoxynibose in the hydnolysate was measured by amodification of the procedure of Giles and Myers (7). To 0.5ml of the DNA hydnolysate, 0.5 ml of 4% diphenylamine inglacial acetic acid and 0.025 ml of 0.2% aqueous acetaldehyde were added . The samples were incubated at 35°for 18hr and then read in the spectrophotometer as previouslydescribed(7).

RESULTS

Development of Hyperplasia. The uncultured bladder epithelium showed a low frequency of [3H]TdR-labeled nuclei(<0.1%). When bladder was cultured in Ham's medium, thiscondition persisted for the 1st 24 hr. After this period, alange increase was observed in the frequency of labeledcells (Chart 1). This trend continued until 4 days of cultureand then declined. At 10 days of culture, however, thefrequency of cells undergoing DNA synthesis was still considenably above that of normal bladder.

Cells undergoing DNA replication were also scored ac

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Chart 1. Time course of [3H]TdR incorporation into DNA of bladder epitheha cultured in Ham's Medium F12. Each point represents the mean percentage of labeled epithelial cells from 4 on 5 cultures ±SE. All tissues werelabeledfor1 hr.

1422 CANCERRESEARCHVOL. 37

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Induction and Control of Bladder Hyperplasia

cording to whether they were basal or intermediate cells. At2 days of culture, greater than 80% of the labeled cells wereclassified as basal cells. At 3 days, label was nearly equallydistributed between intermediate and basal cells (Chart 2).By 4 and 5 days, however, intermediate cells were thepredominant cells engaged in DNA synthesis. This patternsubsequently reversed, and by 10 days o@culture basal cellswere again the predominant DNA-synthesizingcells. Only 2labeled superficial cells were encountered in the entire expeniment.

That the DNA synthesis which was observed during theculture period was followed by cell division is shown inChart 3 by the pattern of increase in the frequency of mitosis. Mitotic figures were not seen until the 2nd day of cultureat which time 2 were observed out of a total of 2527 countedcells. The increase in frequency of mitotic figures followed,by a slight lag, the increase in labeled cells. The highestfrequency of mitotic figures was observed at 3 and 4 days ofculture, after which the frequency declined. As was the casefor cell labeling, the frequency of mitotic figures at 10 days

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Chart 4. Time course of increase in nuclear number in bladder epitheliumcultured in Ham's Medium F12. Each point represents the mean number ofnuclei pen unit length (150 @m)of epithelia from 4 on 5 cultures ±SE.

of culture remained considerably above that of the uncultuned bladder.

It is evident from the histology that the increases in DNAsynthesis and mitosis resulted in more epithelial cells (Figs.2 to 4). It was also possible to obtain a quantitative estimation of increased cell number during the culture period bycounting the number of nuclei pen flat unit length of epithehum (Chart 4). Cell number was essentially constant for the1st 2 days. Between 2 and 7 days of culture, the number ofcells increased linearly. The magnitude of increase in nucleiper unit length of epithelium between 2 and 10 days wasapproximately 2.6-fold.

Inhibition of Hyperplasia with Hydrocortisone. Hydrocontisone has been shown to inhibit the DNA synthesis andgrowth of some cells (2, 6, 11, 22). Having established thefeatures of the hyperplastic response in F12 medium, theability of hydrocortisone to control this hyperplasia wasexamined. In bladders cultured continuously for 3 days inthe presence of various concentrations of hydrocortisone,the incorporation of [3H]TdR into DNA was strongly inhibited at concentrations of 2.1 and 21 @M(Chart 5). A smalldegree of inhibition was observed at 0.21 @M.

It was a consistent observation that the sensitivity of[3H]TdR incorporation to hydrocortisone depended uponthe day of culture on which the tissues were labeled. Cellssynthesizing DNA at 2 days of culture were considerablyless sensitive to hydrocortisone than those synthesizingDNA at 4 days (Table 1). DNA-replicating cells at 2 days ofculture showed essentially no inhibition of [3H]TdR incorporation into DNA by 0.21 @Mhydnocortisone, whereas thosecellsreplicatingat4 dayswere inhibitedby approximately60% with the same concentration. At a 10-fold higher concentration, cells replicating DNA at 2 days showed a greaten

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Chart 2. Relative [3H]TdR labeling of basal and intermediate cells of bladder epithelium cultured in Ham's Medium F12. Each point represents themean percentage of labeled epithelial cells from 4 or 5 cultures. All tissueswere labeled for 1 hr.

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Chart 3. Time course of the occurrence of mitotic figures in bladderepithelium cultured in Ham's Medium F12. Each point represents the meanpercentage of mitotic figures in epithelia from 4 or 5 cultures ±S.E.

1423MAY 1977

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HC CONCENTRATION(tiM)

Chart 5. Effect of different hydrocortisone (HC) concentrations on I3HITdRincorporation into DNA of bladder epithelium cultured in Ham's Medium F12.Tissues were cultured for 3 days continuously in the presence of the mdicated hydrocortisone concentrations and then labeled for the last 3 hr of theculture period with 5 @Ciof [3H]TdR per ml of medium. Eachpoint representsthe mean cpm pen g.@gDNA from 5 cultures ±S.E. Shaded area, mean cpmper @.tgDNA ±S.E. from 5 half-bladders labeled for 3 hr immediately aftersurgical removal.

degree of inhibition than at the lower concentration, but theinhibitionwas stilllessthanthatobservedin4-dayneplicating cells. That the inhibition of [3H]TdR incorporation intoDNA was due primarily to the inhibition of DNA synthesis isshown by the suppression of epithelial hyperplasia (Chart6). Hydnocortisone present continuously in the medium for7 days at a concentration of 2.1 @Mcaused a 75% inhibition

of epithelial hyperplasia compared to controls. At a 10-foldhigher concentration of hydrocortisone, the inhibition was84%. In addition to this striking suppression of hyperplasia,epithelia cultured in the presence of hydnocortisone (Fig. 6)did not display the atypia characteristic of epithelia culturedin the absence of hydrocortisone (Fig. 5). Epithelial cellsalso displayed an apparent increase in cytoplasmic area asa result of hydrocortisone treatment. No evidence of epithehal degeneration on other manifestations of toxicity to hydrocortisone was observed.

DISCUSSION

The transitional epithelium is not a rapidly renewing tissue (10). However, as the result of some conditions, such asexposure to carcinogens on infection, it displays a strikingcapacity for proliferation and growth (14). It has been demonstrated in this study that culture in Ham's Medium F12 isanother condition that results in marked proliferative activity and growth in the transitional epithelium. Although theperiod of intense proliferation is of relatively brief durationin Medium F12, it should be emphasized that cell prolifenation continues throughout the duration of the culture pe

Hydrocortisone inhibition of (3HJTdR incorporation into DNAofbladderepithelial cells as a function of time incultureHalf-bladders

were cultured in Ham's Medium F12 for 2 or 4daysandlabeled for the last 3 hr of the culture period with[3H]TdR.Inhibition

was calculated from the mean specific activity of 4 or5half-bladdersand is expressed as the percentage inhibitionof[3H]TdR

incorporation into DNA relativeto control cultureswhichwerenot exposedto hydrocortisone.%

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D. H. Reese et al.

18 nod. This is in contrast to some chemically defined culturesystems in which only a limited period of proliferation issupported(17).

The absence of mitotic activity before the appearance of[3H]TdR incorporation into DNA indicates that the transitional epithelium of the rat bladder is composed primarily ofcells that are in the G, phase of the cell cycle. It is possible,however, that division by a small population of G2 cellscould have occurred during the 1st 12 hr of culture and wasnot detected.

The time sequence of the appearance of [3H]TdR-Iabeledcells in the basal and intermediate layers of the epitheliumsuggests that hyperplasia begins in basal cells. Labeling issubsequently seen in intermediate cells, but it is not possible to determine from this study whether this labeling is inâ€â€˜original― intermediate cells or in “daughter― basal cells

that have migrated into the intermediate zone after division.

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Chart 6. Inhibition of hyperplasia with hydnocortisone. Tissues were cultuned in Ham's F12 medium continuously for 7 days in the absence ofhydrocortisone (Control) on at hydrocortisone concentrations of 2.1 or [email protected] tissues were not cultured (0 Day). These were fixed immediately

after removal from the animal and indicate initial values. Data are expressedas the mean number of nuclei per unit length (150 @m)of epithelium from 5cultures±SE.

1424 CANCER RESEARCH VOL. 37

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Induction and Control of Bladder Hyperplasia

It is possible that some labeled cells were originally intermediate cells, since it has been shown in the mouse bladderthat all 3 types of transitional epithelial cells (basal, intenmediate, and superficial) retain the capacity for proliferation(10, 24).

Since the highest frequency of [3H]TdR-labeled cells occurredat4 days ofculture,itwould be expectedthatthepeak in mitotic acitivity would occur subsequent to 4 days.This was not the case; mitotic activity peaked at 3 to 4 days.To resolve this discrepancy, a more detailed analysis of thecell-cycle kinetics in the various cell populations of organcultured transitional epithelium will be required.

It has been suggested recently that some tissues, which

normally do not undergo rapid renewal but have retainedthe capacity for rapid growth, are particularly sensitive tothe hydrocortisone suppression of [3H]TdR incorporationinto DNA (12). We have demonstrated in the present studythat the transitional epithelium of the urinary bladder can beadded to this class of tissues and that this suppression isprobably due exclusively to the suppression of DNA synthesis.

The temporal change in hydrocortisone sensitivity in cultune is of particular interest. Epithelial cells synthesizingDNA at 2 days are considerably less sensitive to hydnocortisone than those synthesizing DNA at 4 days. Since thepredominant DNA-synthesizing cells at 2 and 4 days arebasal and intermediate cells, respectively, the data suggestthat intermediate cells may be considerably more sensitiveto hydrocortisone. Whether this difference in sensitivity meflects some general change in the epithelium such as increased permeability to hydrocortisone or whether it mdicates an actual difference in responsiveness of the 2 celltypes will require more study. In connection with this latterpossibility, it has been shown in squamous epithelial cellsof the mouse forestomach that a period exists (called “Rphase―)during the last 6 to 9 hr of G1when cells becomeinsensitive to hydrocortisone and thus can no longer bestopped from eventually entering S phase (6). Since thesequence of events described in this paper indicates thatthe basal cells ofthe transitional epithelium are also in G,, itis possible that these cells may accumulate at, or just before, a point in G1which is similar to “R phase.―

Insulin was routinely included as a culture medium component in the hydrocortisone experiments because it hasbeen shown that insulin and hydrocortisone display synergistic action in some culture systems (15, 23). Recent evidence (0. H. Reese and R. D. Friedman, unpublished observation) shows that insulin alone has no apparent effect onthe hyperplastic response of bladder epithelium in MediumF12and thatinsulinisnotnecessaryforthehydrocortisoneinhibition of this hyperplasia.

The concentration of hydrocortisone necessary to inhibitDNA synthesis in the present study is at least 10 timeshigher than that required to inhibit DNA synthesis in someother epithelial cells (11). Whether this greater requirementfor hydrocortisone is a characteristic of the transitionalepithelium, organ culture, serum-free medium, on someunknown entity will require further study.

1425MAY 1977

ACKNOWLEDGMENTS

We thank Dr. E. Brad Thompson, for helpful suggestions in the preparation of this manuscript; and The Upjohn Co., Kalamazoo, Mich. , and Eli Lillyand Co., Indianapolis, Ind., for generous gifts of hydrocortisone hemisuccinate and insulin, respectively. Doris Little has provided expert secretarialassistance.

REFERENCES

1. Cooper, P. H., Waisman, J., Johnston, W. H., and Skinner, D. G. SevereAtypia of Transitional Epithelium and Carcinoma of the Urinary Bladder.Cancer, 31: 1055-1060, 1973.

2. Cox, R. P., and Macleod, C. M. Alkaline Phosphatase Content and theEffects of Prednisolone on Mammalian Cells in Culture. J. Gen. Physiol.,45:439-485,1962.

3. Dulbecco, R., and vogt, M. Plaque Formation and Isolation of Pure Lineswith Poliomyelitis Viruses. J. Exptl. Med., 99: 167-182, 1954.

4. Eisenberg, R. B., Roth, R. B., and Schweinsbeng, M. H. Bladder Tumorsand Associated Proliferative Lesions. J. Urol., 84: 544-550, 1960.

5. Fannow,G. M., Utz, D. C., and Rife, C. C. Morphological and ClinicalObservations of 21 Patients with Early Bladder Cancer Treated with TotalCystectomy. Cancer Res., 36: 2495-2501 , 1976.

6. Frankfurt, 0. 5. Effect of Hydrocortisone. Adrenalin and Actinomycin Don Transition of Cells to the DNA Synthesis Phase. Exptl. Cell Res., 52:222-232, 1968.

7. Giles, K. W. , and Myers, A. An Improved Diphenylamine Method for theEstimation of Deoxyribonucleic Acid. Nature, 206: 93, 1965.

8. Ham, R. G. Clonal Growth of Mammalian Cells in a Chemically Defined,Synthetic Medium. Proc. NatI. Acad. Sci. U. S., 53: 288-293, 1965.

9. Koss, L. G., Tiamson, F. M., and Robbins, M. A. Mapping Cancerous andPrecancerous Bladder Changes: A Study of the Urotheium in Ten Surgically Removed Bladders. J. Am. Med. Assoc., 227: 281-286, 1974.

10. Levi, P. E., Cowen, D. M., and Cooper, E. H. Induction of Cell Proliferation in the Mouse Bladder by 4-Ethylsulphonyl-Naphthalene-1-Sulphonamide. Cell Tissue Kinet., 2: 249-262, 1969.

11. Loeb, J. N., Borek, C., and Yeung, L. L. Suppression of DNA Synthesis inHepatoma Cells Exposed to Glucocorticoid Hormone in Vitro. Proc. NatI.Acad. Sci. U. S., 70: 3852-3856, 1973.

12. Loeb, J. N., and Yeung, L. L. Effects of Cortisone on Thymidine Incorponation by Various Nonlymphoid Tissues of the Weanling Rat. Proc. Soc.Exptl. Biol. Med., 143: 502-507, 1973.

13. Melicow, M. M. Histological Study of Vesical Urothelium Interveningbetween Gross Neoplasms in Total Cystectomy. J. Urol., 68: 261-279,1952.

14. Mostofi, F. K. Potentialities of Bladder Epithelium. J. Urol., 71: 705—714,1954.

15. Reed, G. B., and Gnisham, J. W. Insulin and Hydrocortisone Effects onViability and Glycogen Stores of Postnatal Rat Liver in Organ Culture.Lab. Invest., 33: 298-304, 1975.

16. Reese, D. H., Friedman, R. D., Smith, J. M., and Sporn, M. B. OrganCulture of Normal and Carcinogen-treated Rat Bladder. Cancer Res., 36:2525-2527, 1976.

17. Rubin, H. Nonspecific Nature of the Stimulus to DNA Synthesis in Cultures of Chick Embryo Cells. Proc. NatI. Acad. Sci. U. S., 72: 1676—1680,1975.

18. Schade, R. 0. K., and Swmnney,J. Precancerous Changes in BladderEpithelium. Lancet, 2: 943—946,1968.

19. Schade, R. 0. K., and Swinney, J. The Association of Unothelial Atypismwith Neoplasia: Its Importance in Treatment and Prognosis. J. Urol., 109:619—622,1973.

20. Simon, W., Condonnier, J. J., and Snodgrass, W. T. The Pathogenesis ofBladder Carcinoma. J. Unol., 88: 797—802,1962.

21. Skinner, D. G., Richie, J. P., Cooper, P. H., Waisman, J., and Kaufman,J. J. The Clinical Significance of Carcinoma in Situ of the Bladder and ItsAssociation with Overt Cancer. J. Unol., 112: 68-71 , 1974.

22. Smith, B. T., Torday, J. S., and Ginoud, C. J. P. Evidence for DifferentGestation-Dependent Effects of Cortisol on Cultured Fetal Lung Cells. J.Clin. Invest., 53: 1518-1526, 1974.

23. Topper, Y. J., and Oka, T. 5teroids and the Development of MammaryEpithelial Cells. In: B. R. Rabin and R. B. Friedman (eds.), Effects ofDrugs on Cellular Control Mechanisms, pp. 131-150. London: MacMillan, 1971.

24. Walker, B. E. Radioautographic Observations on Regeneration of Transitional Epithelium. Texas Rept. Biol. Med., 17: 375-384, 1959.

25. VVaymouth, C. Rapid Proliferation of Sublines of NCTC Clone 929 (StrainL) Mouse Cells in a Simple Chemically Defined Medium (MB752/1). J.NatI. Cancer Inst., 22: 1003-1017, 1959.

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Fig. 1. A bladder explant cultured for 7 days in Ham's Medium F12. One-half of the epithelium was scraped away from the explant (see “MaterialsandMethods―).The explant was then fixed for histology. The plane of the histological section was made at right angles to the plane of scraping so that the photoshows adjacent scraped and unscraped areas of the epithelium. Note the complete removal of epithelium from the scraped portion without apparent damageto submucosal tissues. H & E, x 145.

Fig. 2. Autonadiograph of a bladder explant labeled for 1 hr with [3H]TdR immediately after surgical removal from the animal. Hematoxylin-phloxine B, x360.

Fig. 3. Autoradiograph of a bladder explant cultured in Ham's Medium F12 for 4 days before [3H]TdR labeling for 1 hr. Hematoxylin-phloxine B, X 360.Fig. 4. Autonadiognaph of a bladder explant cultured in Ham's Medium F12 for 10 days before (3HITdRlabeling for 1 hr. Hematoxylin-phloxine B, x 360.

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Fig. 5. Area of an epithelial downgrowth in a bladder explant cultured for 7 days in Ham's Medium F12. This explant is from the same experiment as tissueshown in Fig. 6. Note the extensive dysplasia. H & E, x 360.

Fig. 6. Bladder explant cultured for 7 days in Ham's Medium F12 in the presence of 21 @tMhydrocortisone. Note the normal transitional morphology of theepithelium. H & E, x 360.

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1977;37:1421-1427. Cancer Res   David H. Reese, Rosalind D. Friedman and Michael B. Sporn  in Organ-cultured Rat Urinary BladderInduction of Hyperplasia and Its Suppression by Hydrocortisone

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