[CANCER RESEARCH 37, 2257-2261, July 1977]

Stimulation of DMA Synthesis by Human Placental Lactogen orInsulin in Organ Cultures of Benign Human Breast Tumors

Clifford W. Welsch1 and M. Jean McManus

Department ot Anatomy, Michigan State University, East Lansing, Michigan 48824

SUMMARY

Twenty biopsy specimens of benign human breast tumorsobtained from 20 patients were processed into small slicesand individually cultured for 2 days in Medium 199. Themedium was supplemented with bovine insulin (5.0 ¿tg/ml),human placental lactogen (HPL) (10.0 /^g/ml), or ovineprolactin (10.0 /ng/ml). Four hr prior to termination,[3H]thymidine was added to the culture medium to deter

mine DMA synthesis. The addition of insulin to the culturemedium consistently increased: (a) the mean incorporationof [3H]thymidine into chemically extracted DNA (p < 0.05);(o) the mean number of [3H]thymidine-radiolabeled epithelial cells (p < 0.05), and (c) the mean number of epithelialcells bearing mitotic figures. The addition of HPL increasedthe mean number of [3H]thymidine-radiolabeled epithelial

cells (p < 0.05) and the mean number of epithelial cellsbearing mitotic figures (p < 0.05). [3H]Thymidine incorpora

tion into chemically extracted DNA was also increased whenHPL was added to the medium, although this increase didnot quite achieve the 5% level of significance. The additionof ovine prolactin to the culture medium did not have anysignificant effect on DNA synthesis. This study providesevidence that insulin and HPL are direct stimulants of DNAsynthesis of the epithelium contained in benign humanbreast tumors.

INTRODUCTION

In recent years, there has been a surge of interest inpituitary prolactin as a hormone that may be important inhuman breast tumorigenesis. This renewed interest hasevolved largely because of the recent availability of sensitiveassays for this hormone (14) and its membrane receptor (13,23). Results showing that pituitary prolactin is a key hormonal factor in murine mammary tumorigenesis have alsomarkedly served to focus attention on this peptide as apotentially critical hormonal agent in oncogenesis of thehuman breast (for review, see Ref. 31).

Although prolactin has been shown to stimulate growth ofnormal and tumorous rodent mammary tissue, in vivo (19,21, 24, 29) and in vitro (6, 8, 20, 32), it has not yet beenestablished that this hormone is mitogenic to human breasttumors. Organ culture techniques provide one means

' Recipient of Research Grant BC-220C from the American Cancer Societyand Grant CA-13777 from the USPHS, NIH. USPHS, National Cancer Institute Research Career Development Awardee, CA-35027.

Received January 18, 1977; accepted April 19, 1977.

whereby the direct effects of hormonal agents on growthand differentiation of human breast tissues can be determined. It is the purpose of this study, therefore, to determine whether or not prolactin is mitogenic to benign humanbreast tumors maintained in organ culture.

Two hormones known for their mammotrophic effects inrodents, HPL2 (12, 33) [which ¡schemically similarto humanpituitary prolactin and growth hormone (3,15)], and OPR (8,24), have been used as prolactin sources in these studies.The mitogenic effects of these hormones are compared withthose of bovine insulin, a peptide previously reported topromote in vitro growth of both rodent (9,11, 20, 28, 32) andhuman (5, 10, 28) mammary epithelium.

MATERIALS AND METHODS

Twenty human benign breast tumor biopsy specimens,obtained from 20 premenopausal patients, were placed in achilled holding medium (Medium 199, Hanks' base, con

taining 50.0 ID of penicillin G per ml) and returned to thelaboratory within 30 min. The biopsy specimens were immediately and carefully trimmed of adipose tissue while immersed in the holding medium. All tissue preparations wereperformed in a laminar flow hood under aseptic conditions.

Preparation of Slices for Organ Culture. Slices of biopsyspecimens were prepared with the aid of a Stadie-Riggstissue slicer and a No. 10 Bard-Parker surgical blade. Eachbiopsy specimen provided 5 to 15 large slices ranging from10 to 15 mm in diameter and 0.1 to 0.3 mm thick. Each slicewas processed by a series of halvings with a surgical blade,i.e., each half being subsequently halved until the slicesmeasured approximately 1 x 1 mm. The small slices (1 x 1mm) were pooled, then placed in 10- x 30-mm Falcon disposable Petri dishes, 10 slices/dish. In addition, a singlelarger slice (3x3 mm) was added to each small Petri dish.Each Petri dish contained 2.0 ml of the culture medium.

Each biopsy specimen was divided into 3 groups, i.e., acontrol and 2 experimental groups. Each group (controlsand experimental) had 9 small Petri dishes containing atotal of 90 small slices and 3 (for biopsy Specimens 11 to 20)or 9 (for biopsy Specimens 1 to 10) larger slices. The smallPetri dishes were placed in a covered water-saturated largerFalcon disposable Petri dish (15 x 100 mm), 3 small dishesper larger dish. These Petri dishes were then placed in asmall gassing chamber and housed in an incubator at 37°.The chambers were continuously infused with gas (95% O2-

2 The abbreviations used are: HPL, human placental lactogen; OPR, ovine

prolactin.

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5% CO2) during the culture period. All biopsy specimenswere individually cultured; slices from different specimenswere never combined. The large number of randomly selected small slices per group provides reasonable assurance that an equal quantity of epithelium is distributedamong the 3 groups at the onset of culture.

The culture medium used in these studies was Medium199, modified Earle's salts (1250 mg NaHCO., per liter) ob

tained from Grand Island Biological Co., Grand Island, N. Y.The hormones used in this study and their concentrations inthe culture media were: OPR (NIH-S-9, 27.0 ID/mg) (10.0

/Lig/ml), human placental lactogen (Nutritional BiochemicalCorp., Cleveland, Ohio) (10.0 /¿g/ml), and bovine pancreasinsulin (California Biochemical Corp., La Jolla, Calif., 22.5ID/mg) (5.0 /ug/ml). All media contained penicillin G (Nutritional Biochemical Corp.) (50 ID/ml). After all additions, themedia were passed through a Millipore filter (0.45 p.m),added to the Petri dishes, and the entire culture assemblywas frozen (-20°) until the biopsy specimens were brought

to the laboratory (within 1 month).At the end of the 2nd day of culture, 4 hr prior to termina

tion, sterile [merr)y/-3H]thymidine (New England Nuclear,

Boston, Mass., 40 to 60 Ci/mmole) was added to the culturemedium at a concentration of 1.125 ^Ci/ml. Termination ofthe cultures was designed to facilitate quick removal of thesmall slices from the media in order to obtain a wet weightfor each group, and then storage in 0.9% NaCI at -20° for

DNA extraction and analysis. The larger slices were alsoquickly removed, fixed in Bouin's fluid, and stored for ra-

dioautographic [3H]thymidine and histological (mitotic fig

ures) analysis.DNA Extraction and Analyses of Cultured Slices. For

DNA extraction and analysis, the tissues from each groupwere ground in 0.9% NaCI solution with a Willems Polytronhomogenizer. An equal volume of 20% trichloroacetic acidwas added to the homogenate; the resulting precipitate wascentrif uged (3000 x g) and washed twice with 10% trichloroacetic acid. The precipitate was then washed twice in sodium acetate and in methanol and in chloroform-methanol,

once in 100% ethanol, and once in 100% ethyl ether, in thatorder, to remove lipid and H2O. In all of the foregoing procedures, the preparations were kept constantly cold. Thedefatted-dehydrated extract was placed in a ventilated fume

hood (12 to 18 hr), then in a vacuum desiccator (24 hr), andwas subsequently weighed.

The defatted-dehydrated extract was digested (3 hr, 37°)

with repeated stirrings in 0.3 N KOH. The preparation wascooled, precipitated with cold 10% perchloric acid, centri-

fuged (3000 x g), and washed twice. The precipitate wasthen incubated for 30 min with constant stirring in hot (70°)

5% perchloric acid in which the DNA was soluble. Thispreparation was cooled, centrif uged (3000 xg), and washedtwice with cold 5% perchloric acid. The supernatant wascollected for DNA and [3H]thymidine analysis. DNA contentwas quantitatively determined (in duplicate) by the diphen-ylamine-colorimetric method of Burton (4). Calf thymus

DNA (Sigma Chemical Co., St. Louis, Mo.) was used as astandard. The [3H]thymidine content was determined by

pipetting neutralized aliquots (in triplicate) of the supernatant onto 2.3-cm Whatman No. 3 filter papers. The filterpaper was air-dried and placed in a liquid scintillation vial

containing toluene PPO-POPOP fluors. The samples werecounted in a Beckman LS-100C liquid scintillation counter

with a counting efficiency of 56%. Counting efficiency wasdetermined by the percentage counts of a precisely calibrated (dpm), sealed tritium standard. The results wereexpressed as cpm [3H]thymidine per /¿gof DNA. Signifi

cance of differences between mean cprn/Vg DNA values ofeach group was analyzed by the t test for paired observations.

Radioautographic [3H]Thymidine and Mitotic Figure

Analyses of Cultured Slices. For radioautographic analyses, the fixed slices (9 slices/group for Biopsies 1 to 10, 3slices/group for Biopsies 11 to 20) were embedded in paraffin. Each slice was processed into 6 paraffin sections (5 to 7¿imthick), and placed on microscopic slides. The slideswere coated with a standard emulsion (Kodak NTB-2) andstored at 4°for 3 weeks. They were developed, fixed, and

stained (hematoxylin and eosin) by a standard procedure(25). Only cells bearing >4 silver grains were scored. Thetotal number of radiolabeled epithelial cells was determinedin each section.

For mitotic figure analyses, paraffin sections adjacent tothose used for radioautographic analyses were stained withhematoxylin and eosin. Total number of mitotic figureswere determined in each section. Significance of differences between mean number of mitotic figures of eachgroup and mean number of radiolabeled cells of each groupwas analyzed by the f test for paired observations.

RESULTS

The addition of insulin to culture medium containingslices of human breast tumors resulted in a mean increasein: (a) incorporation of [3H]thymidine into chemically extracted DNA (p < 0.05); (b) number of [3H]thymidine-radio-

labeled epithelial cells (p < 0.05), and (c) number of epithelial cells bearing mitotic figures (Tables 1 and 2). Nineteenof the 20 breast biopsies showed an increase in the incorporation of [3H]thymidine into chemically extracted DNA when

insulin was added to the culture medium. Of the 15 culturedbreast biopsy specimens that were microscopically examined for [3H]thymidine-radiolabeled epithelial cells, all

showed an increase in the number of labeled cells wheninsulin was added to the culture medium. Of the 18 culturedbiopsy specimens examined for mitotic figures, 15 showedan increase in the total number of epithelial cells bearingmitotic figures after treatment with insulin; 3 showed nomitotic figures, regardless of whether insulin was added tothe culture medium.

The addition of HPL to culture medium containing slicesof human breast tumors resulted in a significant (p < 0.05)mean increase in the total number of: (a) [3H]thymidine-

radiolabeled epithelial cells and (b) epithelial cells bearingmitotic figures (Table 1). [3H]Thymidine incorporation into

chemically extracted DNA was also increased when HPLwas added to the medium, although this increase did notquite achieve the 5% level of significance (p < 0.10) (Table1). Seven of the 10 cultured breast biopsy specimensshowed a substantial increase in the incorporation of[3H]thymidine into chemically extracted DNA and in the

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Hormones and Breast Tumors in Vitro

Table 1Effect of HPL and insulin on DNA synthesis of 2-day organ cultures of human breast tumors

3H]Thymidine/Aig DMA" (cpm) Total no of [3H]thymidine-labeledcells'" (radioautographs)

Total no. of cells in mitosis'(mitotic figures)

Biopsyspecimen"12345678910Controls40.930.991.7155.954.542.9126.3496.8112.035.5Insulin93.5100.8234.3258.8119.4105.5159.3702.961.5136.2HPL71.963.2203.492.475.863.3126.9704.191.488.8Controls2350614690411296159793Insulin63692118333475589614161981HPL496312933335120335517411174Controls950104101208Insulin31413721571346313HPL222244185123242

Mean 118.7" 197.3' 158.1' 759° 2410" 2009' 8 29 19*

" Ten benign breast biopsy specimens were obtained from 10 premenopausal patients. Biopsy Specimens 1, 2, 6,and 10 were evaluated histopathologically as fibroadenomas; Specimens 4, 5, and 7 to 9, as fibrocystic disease; andSpecimen 3, as both fibroadenoma and fibrocystic disease.

* Slices (270; 1x1 mm) were analyzed for each biopsy specimen (90 slices/group). The slices from each group werepooled and analyzed for [3H]thymidine incorporation into DNA.

r Slices (27; 3x3 mm) were analyzed for each biopsy specimen (9 slices/group). Each slice was processed into 6sections and the total number of labeled cells or mitotic figures were determined for each group (54 sections/group).

"•'•'p < 0.05, d/e; e/f.'•"•<p< 0.05, g/h; gli.>-kp < 0.05, j/k.

Table 2Effect of OPfíand insulin on DNA synthesis of 2-day organ cultures of human breast tumors

Biopsy speci- [3H]Thymidine/^g DNA»(cpm)

men0n121314151617181920Controls78.316.924.16.18.234.812.728.876.429.1Insulin161.828.527.38.620.759.234.8216.494.238.5OPR125.816.119.35.59.935.642.943.963.338.3Total

no. of [3H]thymidine-la-beled cells''(radioautographs)Controls3745357100850079125130Insulin16075124319451477154191094OPR0535455469655081559Total

no. of cells in mitosis'(mitoticfigures)Controls00520112410Insulin001434326880OPR001114142500

Mean 31.5 69.0 40.1 377" 932' 336' 8 19°Ten benign breast biopsy specimens were obtained from 10 premenopausal patients. Biopsy Specimens 11 and 17

to 19 were evaluated histopathologically as fibroadenomas; Specimens 12, 14, 16, and 20 as fibrocystic disease;Specimen 15 as both fibroadenoma and fibrocystic disease; and Specimen 13 as virginal hypertrophy (patient was 15years old).

6 Slices (270; 1 x 1 mm) were analyzed for each biopsy specimen (90 slices/group). The slices from each group werepooled and analyzed for [3H]thymidine incorporation into DNA.

' Slices (9; 3 x 3 mm) were analyzed for each biopsy specimen (3 slices/group). Each slice was processed into 6sections, and the total number of labeled cells or mitotic figures were determined for each group (18 sections/group).

"•'•'p< 0.05, d/e, elf.

number of cells bearing mitotic figures when HPL wasadded to the culture medium. Seven cultured breast biopsyspecimens were examined microscopically for [3H]thymi-dine-labeled epithelial cells, and all 7 showed increasednumbers of labeled cells in the HPL-treated group, compared with controls. However, HPL was generally less effective as a DNA synthesis stimulant than was insulin. Theaddition of OPR to culture medium containing slices of

human breast tumors did nof significantly influence themean incorporation of [3H]thymidine into DNA, the meantotal number of [3H]thymidine-radiolabeled cells or themean total number of cells bearing mitotic figures (Table 2).

The radioautograph and mitotic figure results in thisstudy are presented as total number of cells rather than asthe ratio of these cells to a given number of epithelial cells,the more customary procedure. Because of the heterogene-

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C. W. Welsch and M. J. McManus

ity of benign breast tumors, it is necessary to use a largesampling of the tumor slices, as we have, in order to obtainrepresentative figures for [3H]thymidine-labeled cells. One

cannot count selected areas as is done for more homogeneous tissues. It is therefore necessary to count all the epithelial cells in the tissue sections used for a given group, inorder to present the data as a ratio of a given number ofepithelial cells. This procedure is extremely time consuming. We randomly chose 2 of the cultured breast tumors(Table 1, Biopsy Specimens 2 and 4) and counted the totalnumber of epithelial cells in the sections from which ra-dioautographic counts were obtained. In the sections derived from Biopsy Specimen 2, a total of 22,250 cells wereobserved in the control group, 25,540 cells in the insulingroup, and 20,338 cells in the HPL-treated group. The ratiosof labeled epithelial cells per 1000 epithelial cells were:controls, 23.4; insulin-treated, 86.6; and HPL-treated, 63.6.In the sections of cultured slices from Biopsy Specimen 4, atotal of 5298 cells were observed in the control group, 3462in the insulin-treated group and 6170 in the HPL-treatedgroup. The ratios of labeled epithelial cells per 1000 epithelial cells were: controls, 77.5; insulin-treated, 218.0; andHPL-treated, 195.0. Thus, whether the results are reportedas total numbers of [3H]thymidine-labeled cells or as a ratio,

i.e., labeled cells per 1000 nonlabeled cells, a marked stimulatory effect of insulin and HPL is observed. We thereforedecided that our sampling was representative enough between groups to warrant use of total labeled cells as acriterion of response.

In general, there was an excellent correlation in this studybetween the 3 indices of DMA synthesis, i.e., specific activity of [3H]thymidine into chemically extracted DMA, numberof [3H]thymidine-radiolabeled cells, and number of mitotic

figures. When examining individual cultured biopsy specimens, an enhancement by either insulin or HPL of one ofthese indices almost invariably resulted in an increase inboth of the other indices of DMA synthesis. The morphologyof all 20 of the cultured human breast specimens was wellpreserved; areas of necrosis were rare. Consistent morphological changes of the cultured specimens, as a function ofthe hormonal milieu, however, were not readily apparent.Although, at times, numerous fibroblasts were observed inthe cultured specimens, they were seldom radiolabeled.There was an insufficient quantity of biopsy material fromSpecimens 11 and 12 (Table 2) for radioautographic andhistological analyses. A radioautographic examination wasattempted on Specimens 6 to 8 (Table 1) but, for yet unexplained reasons, these tissues did not process effectivelyand therefore were not available for analysis.

DISCUSSION

DNA synthesis was increased in all 20 of the breast biopsies examined in this study when insulin was added to theculture medium. Only 1 of the 20 biopsy specimens failed toshow a response to insulin of increased incorporation of[3H]thymidine into chemically extracted DNA. This particular biopsy specimen (No. 9) was histologically unique in thatit contained a large quantity of adipose tissue. The adiposetissue may have interfered with the chemical extraction of

DNA; i.e., a portion of the sample may have been lost duringlipid extraction, causing erroneous results. Radioautographic analysis showed that insulin sharply increased thenumber of [3H]thymidine-labeled cells, not only in this particular specimen, but in all the cultured biopsy specimensexamined by this procedure. The biopsy specimen in whichinsulin induced the greatest incorporation of [3H]thymidine

into chemically extracted DNA (No. 18) was a specimenobtained from a patient during pregnancy; all other specimens were derived from nonpregnant patients. It has beenpreviously reported, using strictly morphological criteria,that insulin enhances (5,10) or has no effect (26) on growthof normal ordysplastic human breast tissues maintained inorgan culture. The results of our study provide evidencethat this peptide is consistently stimulatory to DNA synthesis of tumorous (benign) human breast tissues maintainedin short-term organ culture. It is well recognized that thesebiopsy specimens contain varying proportions of diffuselyinfiltrated normal, hyperplastic, and cystic epithelial elements. Studies designed to determine which particulartypes of human breast epithelium (normal and dysplastic)respond to the mitotic stimulus of insulin in vitro are currently in progress in our laboratory.

HPL also substantially increased DNA synthesis of thebreast tumor expiants, although to a lesser degree than didinsulin; OPR was ineffective. These results are in accordwith the data described in a recent brief communication byDilley and Kister (7), indicating that human pituitary prolac-tin or HPL in combination with insulin increased the mitoticindex of organ cultures of normal human breast tissues to alevel higher than that in cultures containing no hormones orinsulin alone; OPR was without a stimulatory effect. It isfairly well established that OPR is an effective mitogen inorgan cultures of normal (6, 8) and carcinomatous (20, 28,32) rat mammary tissues. There are conflicting reports as towhether OPR is stimulatory to cultures of human breastcarcinomas (1,2,18, 22, 28). Rodent mammary tissues havemembrane receptors for OPR (13, 23), whereas humanbreast tissues may be lacking receptors for OPR, as suggested by the recent report of Holdaway and Worsley (16).This would explain the lack of effect of OPR on humanbreast epithelium shown in our study and in that by Dilleyand Kister (7).

HPL was used in our study in lieu of human pituitaryprolactin because the purified human pituitary peptide hasnot been available in sufficient quantities for use in ourorgan culture studies. HPL is structurally slightly more similar to ovine or human growth hormone than it is to OPR orhuman prolactin, although the sequence homologies of theplacental peptide and these pituitary peptides are sufficiently similar to suggest that all of these molecules evolvedfrom a common ancestor (3, 15). Because of the closerstructural similarity of HPL to growth hormone, HPL isfrequently referred to as human chorionic somatomammo-tropin (3). Despite this close structural similarity to thegrowth hormones, the somatotrophic activity of HPL is considerably less than that of growth hormone, having 1.0% ofthe potency of the pituitary peptide (12, 17). On the otherhand, HPL has marked mammotrophic-lactogenic activitiesin lower animals in vivo and in vitro. This placental peptide

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Hormones and Breast Tumors in Vitro

reportedly (12, 33) stimulates pigeon crop sac activity,mammary gland development in pseudopregnant rabbits,mammary gland secretion in mice, and growth of precan-cerous mammary gland lesions in mice (12, 33). It is emphasized that the word "lactogen" in HPL is derived from numerous studies showing that this hormone is mammo-trophic-lactogenic in lower animals; it has not yet beendemonstrated that this hormone is lactogenic in primates(17). The results described in this paper and the earlier briefcommunications by Dilley and Kister (7) are the first directevidence, to our knowledge, that HPL is mammotrophic tohuman breast epithelium.

HPL is secreted by the placental syncytiotrophoblast andhas been detected as early as 12 to 18 days after conception. The level of this hormone then rises progressively untillate pregnancy and then declines slightly (17). The results ofour study provide evidence suggesting that HPL may be afactor in the etiology of dysplastic breast lesions seen inmultiparous women, although these lesions are frequentlyencountered in nulliparous women as well. Although little isknown of the biological activity of human pituitary prolac-tin, it may possess mammotrophic activities similar to HPL(7) and therefore may also play a role in the etiology ofdysplastic (benign and cancerous) breast diseases in nulliparous or nonpregnant women. Prolactin is mitogenic to therat mammary epithelium (6, 8, 24, 29) and may be an essential hormonal factor in mammary tumorigenesis in this species. Indeed, chronic drug-induced suppression of prolac-tin secretion in certain mammary tumor-susceptible miceprevents the development and progression of hyperplasticand neoplastic mammary gland lesions in these animals (27,30). If prolactin can be shown to influence the developmentand growth of human breast tissue, as it does in rodents,then prophylaxis and/or chemotherapeutic control of human breast dysplasias may be possible by acute or chronicdrug-induced prolactin suppression.

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JULY 1977 2261

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1977;37:2257-2261. Cancer Res   Clifford W. Welsch and M. Jean McManus  Insulin in Organ Cultures of Benign Human Breast TumorsStimulation of DNA Synthesis by Human Placental Lactogen or

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