Prolactin isoform 2 as an autocrine growth factor for GH3 cells.

0013.7227/92/1312-0595$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine

Vol. 131, No. 2 Printed in U.S.A.

Society

Prolactin Isoform GH3 Cells* KEVIN AMEAE

A. KROWNt, M. WALKER

YU-FEN

WANG,

2 as an Autocrine TIMOTHY

W. C. HO,

PAUL

Growth A. KELLY,

Division of Biomedical Sciences (K.A.K., Y.-F. W., T. W.C.H., A.M. W.), University Riverside, California 92521-0121; and INSERM (P.A.K.), Unit6 344-Endocrinologie Medecine Necker-Enfants Malades, 75743 Paris Cedex 15, France

of

Factor

for

AND

California, Moleculaire,

Faculte de

to the same 40-50% of the cells and copatched or cocapped along with the receptors. Absence or presence of PRL receptors did not correlate with stage of the cell cycle, as judged by ethidium bromide dual labeling. Cell surface PRL was found to be on PRL-containing cells. These data have fulfilled four criteria necessary for establishment of a substance as a secreted autocrine growth factor: 1) the factor must be secreted; 2) in log growth phase, increased cell proliferation should occur at increased cell densities; 3) the cells must display a receptor for the factor; and 4) there must be a growth response to the factor. Thus we have established that PRL is an autocrine growth factor for at least 40-50% of the GH, cell population. This, to our knowledge, is the first example of autocrine growth factor activity of a major hormone normotopically expressed. (Endocrinology 131: 595-602, 1992)

ABSTRACT Because PRL has growth factor activities in several tissues, we have asked whether it also has autocrine growth factor activity in pituitary GH,, cells. GH, cells were grown at increasing densities in the presence or absence of antirat PRL (polyclonal and monoclonal) or nonspecific antibodies. Cell proliferation increased with increasing cell density, as did the concentration of PRL in the medium. Antirat PRL, but not control antibody, markedly inhibited but did not eliminate cell proliferation, and this effect was diminished with increasing PRL concentration in the medium. PRL receptors were demonstrated on 40-50% of the cells by indirect immunofluorescence using a specific antirat PRL receptor monoclonal antibody. Cell surface PRL was colocalized

F

Materials

OR normal cells to become malignant it is necessary for them to lose growth control and to develop the capacity for invasion of surrounding tissues. In some instances of malignant transformation, these two processes are intimately linked, whereas in others they are clearly separate. We have chosen to study aspects of growth control in a tissue where loss of growth control is a relatively common event, leading to a high prevalence of tumors (l), and where progression to malignancy is rare (2): the anterior pituitary. With this tissue we are therefore able to study one aspect of malignant transformation entirely separately from the other. In many tissues, growth control has been shown to be under the influence of a variety of growth factors and in some instances both positive and negative growth control are involved in the normal maintenance of cell number (e.g. Ref. 3). Many of these growth factors act in an autocrine/ paracrine fashion, and either they or their receptors are overexpressed in transformed cell lines (4, 5). Because it is now well established that PRL can act as a growth factor in several tissues (6-8), we have asked whether PRL acts as an autocrine growth factor within the pituitary itself. First we have asked whether pituitary tumor cells show any growth dependency on secreted PRL.

and Methods

GH, cell culture GH3 cells were obtained from the American Type Culture Collection (Rockville, MD) and have been maintained in the laboratory for the past 5 yr, Routine culture was in Dulbecco’s modified Eagle’s medium containing 4.5 g/liter glucose supplemented with 16% horse serum, 4% fetal bovine serum, nonessential amino acids, 20 U/ml penicillin, and 2 pg/ml streptomycin (DMEM+) (all tissue culture materials from GIBCO Laboratories, Grand Island, NY). Subculture was achieved by trypsinization (0.25% Difco 1:250 trypsin in DMEM), washing, and dilution.

Cell proliferation

studies

GHa cells were plated at various densities in a 200-~1 vol in 96-well microtiter plates in DMEM+ and were cultured for 5 days at 37 C under a water-saturated atmosphere of 5% C02/95% air. Plating density was assessed by hemocytometer counts, and every effort was made to reduce cell clumping to ensure reproducible plating among replicates and accurate counts. To some wells were added either rabbit antirat PRL (IC4; AFP 1753080191281 in DMEM+) to give a final dilution of 1:300, or an equivalent dilution of normal rabbit serum. To a fourth set of wells was added standard rat PRL (rPRL) 86 (AFP 7545 E) to give a final concentration of 5 pg/ml. In an additional set of experiments, cell proliferation was measured in the absence and presence of a monoclonal antibody to ovine I’RL (6Fl1, kindly provided by Dr. Jonathan Scammel, University of South Alabama, Mobile, Alabama) used at a 1:300 dilution of ascites fluid.

Dephosphorylation

Received October 25, 1992. Address all correspondence and requests for reprints to: Dr. Kevin A. Krown, Division of Biomedical Sciences, University of California, Riverside, California 92521-0121. * This work was supported by American Cancer Society Grant BC562 (to A.M.W.) and by NIH Physician Scientist Award HD-00810 (to T.W.C.H.). t Postdoctoral trainee supported by NIH Grant AM-07310.

of

NIDDK

PRL

In each experiment, 15 rg NIDDK PRL (rPRL B6, AFP 7545E) was incubated with 1 U acid phosphatase (human prostate; Sigma, St. Louis, MO) in 0.1 M sodium acetate buffer, pH 5.0, for 1, 2, or 4 h at 37 C. Enzyme-treated or untreated PRL or enzyme alone (all in acetate buffer and after a l-, 2-, or 4-h incubation) were then neutralized, sterilized, and assessed for their affect on GH3 cell proliferation at a concentration of 2 fig/ml.

595

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 23:01 For personal use only. No other uses without permission. . All rights reserved.

596 Measurement

PRL AS AN AUTOCRINE of cell proliferation

RIA The accumulation of PRL in the culture medium of cells plated at different densities was measured in a homologous assay using rabbit antirat PRL IC4 as antibody and rPRL I-5 as standard. iZ51-labeled rPRL was obtained from New England Nuclear-DuPont (Wilmington, DE). This assay has an interassay variation of 15% and an intraassay variation of 7%. It is sensitive to 0.5 rig/ml and shows 0.0001% cross-reactivity with GH.

demonstration

FACTOR

Endo. Voll31.

1992 No 2

Confocal microscopy

After 5 days of culture, a dye calorimetric method, modified from Green et al. (9) was used to measure the number of cells. After removal of the culture medium, 100 pg 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) dye (Sigma) dissolved in 100 ~1 PBS were then added to each well and incubated with the cells at 37 C for 4 h. Only the mitochondria of viable cells are able to sequester the dye and convert it to a purple formazon by dehydrogenase reduction. After removal of the dye solution, the formazon complex was dissolved in 100 ~1 acidified propranol (0.04 N HCl), and an enzyme immunosorbent assay reader (BioRad Laboratories, Richmond, CA) was used to measure the intensity of color development. A standard curve using freshly trypsinized cells to give cell number (assessed by hemocytometer) us. absorbance was always included in the assays of unknown. In preliminary experiments the calorimetric results were cross-checked by Coulter counter (Coulter Electronics, Hialeah, FL) analysis of cell number. Cell viability was assessed by trypan blue exclusion.

Immunofluorescent surface PRL

GROWTH

of PRL receptors and cell

GH, cells were plated onto poly-L-lysine-coated (10) coverslips and then cultured for 2 days in DMEM+. The cells were then rinsed in fresh DMEM without serum, followed by thorough rinsing in Dulbecco’s 0.01 M PBS containing 6% sucrose, pH 7.4 (PBS-6% sucrose; 3 X 5 min). If cell surface localization of PRL was to occur, the rinsing and all steps before fixation were done at 4 C using precooled reagents. After rinsing, cells were placed in a precooled humid chamber where they were first incubated in a presoak solution [3% normal rabbit serum (NRS) in PBS-6% sucrose] for 30 min and then without rinsing incubated for 1 h with rabbit antirat PRL (IC4, I:1000 in PBS-6% sucrose). NRS replaced IC4 in controls, After extensive rinsing with PBS-6% sucrose (3 X 5 min), fluorescein-conjugated sheep antirabbit immunoglobulin G (IgG) 1:50 in PBS; Cappel, Durham, NC) was added, and cells were incubated in the dark for 1 h and then rinsed thoroughly in PBS-6% sucrose. In some experiments, the cells were fixed (see below) before PRL localization both on the cell surface and in the interior of the cell. Some surface PRL-labeled cells were subsequently fixed in periodatelysine-paraformaldehyde containing 6% sucrose (PLP, 11) for 1 h. After fixation, cells were rinsed thoroughly in PBS-6% sucrose and incubated in a presoak solution (3% mouse monoclonal antibody to T-lymphocyte CD4 cell surface antigen (OKT4), 0.4% Triton X-100 in PBS-6% sucrose) for 30 min and then, without rinsing, incubated for 1 h with a mouse monoclonal antibody to a rat liver PRL receptor (IJ,, 5 fig/ml PBS-6% sucrose). OKT4 replaced Ug in controls. After extensive rinsing with PBS-6% sucrose (3 x 5 min), the cells were further incubated for 1 h in Texas red-conjugated goat antimouse IgG (10 fig/ml PBS; Molecular Probes, Eugene, OR) and were then finally rinsed thoroughly in PBS6% sucrose. A control incubation for both PRL receptor and PRL consisted of NRS and OKT4 with both fluorescein-conjugated sheep antirabbit IgG and Texas red-conjugated goat antimouse IgG. As a qualitative assessment of stage of the cell cycle, some cells, fixed and stained for PRL receptor, this time using fluorescein-conjugated goat antimouse IgG (1:50 in PBS; Cappel), were further incubated in RNase A (0.1% in PBS) (Sigma) at 37 C for 30 min followed by incubation in a solution containing 50 pg/ml ethidium bromide, 10 mg/ ml trisodium citrate supplemented with 10 pi/ml 0.5 rnM sodium borohydride solution at room temperature for 15 min. Thorough rinsing in PBS-6% sucrose removed excess ethidium bromide.

Coverslips were mounted onto glass slides with p-phenylene diamine (PPD) [O.l% PPD, 90% glycerol, 10% PBS (12)] to prolong fluorescence necessary for laser scanning microscopic observations. Confocal images were visualized by a laser scanning system (MRC 600; BioRad, Cambridge, MA) coupled to a Nikon (Garden City, NY) Optiphot-2 microscope and using the CM software package (BioRad). The argon laser passes through a blue (488 nm) excitor filter block for fluorescein and a green (514 nm) excitor filter block for Texas red and ethidium bromide. Cells were optically sectioned at 1 to 2-pm intervals to examine the distribution of both PRL and PRL receptors. Cells were photographed using Kodak Tri-X pan ASA 400 film (Eastman Kodak Co., Rochester, NY).

Two-dimensional

gel electrophoresis

Two-dimensional gel electrophoresis of standard PRL B6 preparations and dephosphorylated preparations was performed using a modification of the method of O’Farrell (13). Electrophoresis in the first dimension (isoelectric focusing) was conducted in a 4% acrylamide-0.6% bisacrylamide 3-mm diameter tube gel containing 1.6% (pH range 4-6.5) ampholines (Sigma) and 0.4% (pH 3-10) ampholines for 9000 V-h. Electrophoresis in the second dimension was conducted in a 1.5.mm, 11.5% acrylamide/0.3% bisacrylamide gel containing 0.1% sodium dodecylsulfate. Electrophoresis in this dimension continued for 13 h at a constant current of 10 mA/gel. p1 standards were included at the time of sample preparation in order to unequivocally identify the isoforms. The resultant gels were silver stained (14) and photographed.

Statistical

analysis

For the cell proliferation significance of its difference group by Student’s t test.

studies, each value was assessed for the from the equivalent value in the untreated

Results Cell proliferation

Figure 1 shows a typical standard curve used to calculate cell number from the calorimetric assay. Within certain limits it shows a linear relationship between absorbance,generated in the assay,and cell number, determined by hemocytometer counts. Only optical densities falling within the linear range were converted to cell number when reporting the results of

1.2 1.0 0.8 0.6 0.4 0.2 0.0’

/

’

I

1

4.0 4.2 4.4 4 6 4.8 5 0 5 2 5 4 5 6 5 !’ : ‘1 Lcq

of

Ccl1

NumbEl‘/m!

FIG. 1. A standard curve of GH, cell number measured by the 3-(4.5 dimethylthiazol-2-yl)-2,5diphenyltetrazolium calorimetric dye method. Each point represents the mean + SEM, n = 3. The asterisks mark the upper and lower limits of the assay used in later experiments.


PRL AS AN AUTOCRINE

a-7-ti-5-4-3-2--

I-0

5

10 15 20 25 30 35 40 45 50 55 Initial Density (xf03/m11

30

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27

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15 Inltlal

20 25 40 Density lx103/mll

50

I

6’ 5’ 4’ 3 2 I

0’ 0

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20 Initial

30 Oensity

40

50

GROWTH

591

FACTOR

rate of proliferation up to a maximum at 2.5 X lo4 cells/ml and above. The fold multiplication occurring during the 5 days of culture is presented in Fig. 2B (obliquely hatchedbars). Inclusion of a polyclonal PRL antibody in the culture medium dramatically inhibited proliferation (Fig. 2A, open triangles). The degree of inhibition was greatest at the cell density optimal for proliferation (Fig. 2B, cross-hatching).At higher cell densities, where greater amounts of PRL were secreted (Table l), the degree of inhibition was reduced. When 5-day conditioned medium from cells plated at an initial density of 5 x lo4 cells/ml was substituted for half of the culture medium, antibody inhibition of cells plated at an initial density of 2 x lo4 cells/ml was reduced from the 70.4% shown to 36.2% (data not shown). A 1:300 dilution of antibody is capable of binding about 1 pg/ml PRL. The antibody preparation contained no preservatives and had no effect on cell viability. This result was not duplicated by an equivalent concentration of normal rabbit serum (data not shown). Cross-reactivity of the antiserum with GH was 0.0001% and therefore, given the micrograms per ml concentrations of GH secreted by GH3 cells, was unlikely to be the reason for the inhibitory responseobserved. It is interesting to note that at low cell densities, where the antibody concentration should have been sufficient to bind all the PRL, 6 to 7-fold multiplication still occurred during the 5-day experimental period. When a monoclonal antibody produced against ovine PRL was used, extremely similar results were obtained (Fig. 2C, solid triangles). When rat PRL B6 was added to the culture medium, there was also an inhibition of proliferation. At each density above 2 X lo4 cells/ml this inhibition resulted in the absence of about the samenumber of cells (-200,000 cells) after 5 days (Fig. 2A, open diamonds). Inhibition of proliferation by NIDDK PRL is also evident on Fig. 3 (compare Cl and C2) where the dose is lower and the degree of inhibition is reduced. The isoform composition of the B6 preparation used in these studies is shown in Fig. 4. It was a mixture of isoforms 1, 2, 3, and 3’. It should be noted, however, that different batches of B6 have different isoform compositions (15) and that the actual isoform distribution of a given batch needs to be ascertained at the time of the proliferation assay. Incubation of NIDDK PRL in acid phosphataseresulted in a time-related increase in the growth rate (Fig. 3, compare C2 with El-4) and a decrease in the proportion of the phosphorylated isoforms (data not shown).

60

(xlO’/ml)

FIG. 2. GH, cell proliferation. Proliferation in the absence (0 and 0, A and C; q , B) and presence of a polyclonal PRL-antibody (a, A; B), a monoclonal PRL antibody (A, C), or exogenously added purified rat PRL B6 (0, A; S, B). Error bars are ~SEM, n = 3 or 4. Representative results of three trials.

any given experiment. Asterisksmark the range used in later experiments. Intraassay error was never greater than 15% of the value on the ordinate. As demonstrated in Fig. 2A (open circles) and Fig. 2C (closed circles), plating at increased density, increased the

PRL autoreceptors PRL receptors are demonstrated in Fig. 5. Approximately 40-50% of the cells were positive for PRL receptors (Fig. 5A). Of these, some displayed a diffuse, uniform cell surface TABLE

1. Accumulated

GHa cells/ml” PRL (rig/ml) ’ Initial “n=3. ‘n=4.

numbers

1 x lo4 80 + 15’

PRL

in media 2 x lo4 319 f 21’

after

5 days of culture

4 x lo4 518 -c 44’

of cells.


8 x lo4 663 -r- 31’

PRL AS AN AUTOCRINE

GROWTH

FACTOR

Endo l 1992 Vol131.No2

1 .n 0= n

h \ ‘,

6

~

Cl

c2

El

E2

E4

3. Effect of phosphorylated and dephosphorylated exogenous PRL on GH, cell proliferation. Cells were initially plated at 1.5 X lo*/ ml and were incubated in this case for 3 days rather than the usual 5 days. Cl, Cells plus 1 h buffer-incubated enzyme; C2, cells plus 4 h buffer-incubated standard PRL (corrected for a very small effect of the enzyme alone); El-E4, cells plus standard PRL dephosphorylated for 1, 2, or 4 h. Error bars k SEM, n = 4. C, differs from EP and E,. P < FIG.

0.005and0.05,respectively. I

2

3

31

FIG. 4. Silver-stained two-dimensional gel of purified, exogenously added standard PRL. 1, 2, 3, and 3’ are the isoforms of 24-kD PRL in order of increasing acidity.

fluorescence, some a punctate fluorescence, and some a dense polar fluorescence (Fig. 5B). Using ethidium bromide to illustrate chromatin patterns (Fig. 6), no correlation was found between the absenceor presenceof PRL receptors and stage of the cell cycle. Cells were optically sectioned to verify that the distribution and cell cycle results were not distorted by single sectional plane views. Control incubations showed only ethidium bromide fluorescence. The sametypes of distribution of receptor were seenwith glutaraldehyde fixation. Nuclear fluorescenceon the fluorescein panel in Fig. 6 was due to cross-over in emission spectra between ethidium bromide and fluorescein and should not be interpreted as evidence of nuclear PRL receptors.

FIG. 5. PRL autoreceptors on GH, cells. Low magnification view in A shows 40-50% labeling of cells. o, Unlabeled cells. At higher magnification (B) patching (arrowhead) and capping (double arrowhead) of the receptors is seen. On the cell demonstrating patching, the section plane is equatorial. On the cell demonstrating capping the section grazes part of the outer surface (bar, 5 pm).

showed variously diffuse, punctate, and polar distributions the sameas the receptor and was present on 40-50s of the cells. Receptor fluorescence was absent when Ug was replaced with OKT4 (Fig. 7B), and PRL fluorescence was not found when NRS replaced IC4 (Fig. 7C). No fluorescence was found when OKT4 and NRS and both secondantibodies were used (not shown). When fixed cells were used for PRL localization, optical sectioning through entire cells allowed us to determine that PRL-containing cells also had cell surface PRL (Fig. 8).

Cell surface PRL

Colocalization of cell surface PRL and PRL receptors is shown in Fig. 7A. In this instance, the intense fluorescence of the Texas red tended to obscure the more punctate localization of the receptor, but where there was a specific regional distribution of cell surface PRL and the receptor, they were found colocalized to that region. Cell surface PRL

Discussion

The ability of a particular isoform of 24-kilodalton (kD) PRL to regulate GH3 cell proliferation is demonstrated in this paper. Four criteria for establishing a substanceas an autocrine growth factor have been fulfilled. First, in previous publications (15,16), we have established


PRL AS AN AUTOCRINE

FIG. 6. Dual staining for autoreceptor (left) and chromatin (right). Split images from confocal screen showing fluorescein emission on the left and ethidium bromide emission on the right. Specific receptor antibody used for cells in upper panel. Nonspecific antibody used for cells in lower panel. Note cells with (a) and without (b) PRL receptors in the plane of section in left upper panel and the absence of fluorescence in the control lower left panel. Nuclear fluorescence on left panels was due to cross-over in emission spectra between ethidium bromide and fluorescein. Bars, 10 pM.

that the only form of PRL secreted by GH, cells is PRL isoform 2. Thus, using GH3 cells we can study the activity of a single isoform without the complication of potentially different activities of the other isoforms (see below). The lack of significant PRL storage in GH3 cells under normal culture conditions (15, 17) also tells us that secretion of PRL isoform 2 is predominantly constitutive, i.e. most PRL is released very rapidly after synthesis. We therefore would predict, and it has been demonstrated (18, 19, and results herein), that culture of GH3 cells at increasing densities would result in increasing PRL isoform 2 concentrations in the medium. An increased rate of proliferation at increasing culture densities fulfills a second criterion. It demonstrates the presence of an autocrine factor for proliferation but in no sense directly implicates PRL isoform 2. In fact, autocrine growth responses have been noted previously for GH3 and closely related cells. Thus Danielpour et al. (20) determined that a 2-6 kD molecule was responsible for autocrine growth, and Hinkle and Kinsella (21) that a greater than 50-kD molecule also had autocrine proliferative responses.More recently it appears that this latter molecule may be apotransferrin (22). Other growth factors which have been assessedfor their activity on GH3 cell proliferation include epidermal growth factor (20, 21), basic fibroblast growth factor (20, 23, 24), nerve growth factor (21), GH (21), transforming growth factors-a and -/? (25), insulin-like growth factors 1 and 2 (20), and insulin (20). All of these factors either have no effect or inhibit cell proliferation.

GROWTH

FACTOR

Fulfillment of a third criterion involves demonstration of a receptor for PRL on the GH3 cells. An autoreceptor had been implied by the previous demonstration of [‘251]PRL binding to normal mammotrophs (26) and to estrogen-induced rat pituitary tumors (27), and by our previous studies of the autoregulation of PRL secretion (28). These studies demonstrated the binding of some form of PRL but did not specifically address which isoform/isoforms bound, to what the PRL bound, and, with the exception of our secretion studies, what the functional consequencesof binding were. In this study we have used a monoclonal antibody to the rat liver PRL receptor to demonstrate the presence of a PRL autoreceptor on GHs cells. The monoclonal antibody chosen binds to an epitope distinct from the PRL binding site (29), and hence the antibody can recognize the receptor even in the presence of bound PRL. Recognition of the autoreceptor by the monoclonal antibody establishesthat there is a true PRL receptor present on the GHa cell surface. This receptor may not be identical to the liver receptor, but it at least has one common extracellular region. To answer the question of whether secreted isoform 2 bound. to the autoreceptor, it was necessary to demonstrate cell surface localization of GHJ cell-secreted PRL, and this was achieved. The cells bearing receptors were the same as those bearing cell surface PRL. Coregional distribution of PRL’and receptors suggeststhat the PRL was actually bound to the receptor. The finding of apparent patching and capping of the autoreceptors attested to their mobility in the plane of the membrane and suggestedthat they were truly functional. These patterns are common to descriptions of receptor mobility before ligand internalization (30). These receptor aggregations were not antibody-induced, since some cells that had not been prelabeled for PRL and which were fixed before immunolabeling for receptor showed the same distribution. Additionally, receptor aggregation was not an artifact of fixation, since the same array of patterns was observed whether monovalent or bivalent fixatives were used (data not shown). To demonstrate the functional consequence of PRL isoform 2 binding to its receptor, we interrupted the autocrine loop by incubating the cells in antirat PRL. Interruption of the loop resulted in an inhibition of cell proliferation. The concentration of antibody chosen was designed to bind all of the PRL at low cell densities and, on an equilibrium basis, not all of the PRL at the highest cell densities.Thus we could titrate the effects of the anti-PRL and see a lessening of inhibition at higher cell densities and effectively also do this in the conditioned medium experiment. It is interesting to note that the anti-PRL had its greatest effect at the cell density showing the greatest rate of proliferation. In other words, the antiserum was recognizing the autocrine factor. Duplication of the result with an antibody raised in another speciesand derived from ascitesfluid strongly suggeststhat this effect is PRL-specific and not due to an unknown factor in the antiserum. In addition to thinking about PRL-stimulated growth, one should also note the 6, to 7-fold multiplication of cells at low cell density in the presence of the antibody. This is apparently PRL-independent GH3 cell


600

PRL AS AN AUTOCRINE

GROWTH

FACTOR

Endo Vol131.

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1992 No 2

growth. This could be growth in response to other autocrine factors discussed above (20-22) and/or it could be growth of the non-PRL-responsive cells in the population. Only between 40-50s of the cells were positive for autoreceptor and cell surface PRL. That the lack, both of an autoreceptor and cell surface PRL, was not simply a function of cell cyclic display of receptors was demonstrated in the ethidium bromide double-labeling experiments. These experiments showed no correlation between the absence of labeling and stage of the cell cycle as assessed by chromatin patterns. Although more subtle changes in receptor number, distribution, or affinity may well occur during the cell cycle of receptor-positive cells, at least the absence of receptor could not be ascribed to this cause. Heterogeneity among GH, cells has been described in terms of PRL storage (31, 32). Recent studies (33) have indicated that a GH, cell may secrete PRL and/or GH, with its preferential product dependent on the hormonal environment. The absence of surface PRL on some cells may indicate that they are only secreting GH. It has been suggested that a shift of PRL to GH secretors or vice versa may be due to a direct interconversion of cell types (34) and that the dual (PRL/GH) secretor represents a transitional state of cell conversion. It is therefore possible that under circumstances where PRL isoform 2 secretion is high, there is preferential proliferation of one secreting type over another. Because we have not quantitatively determined that all PRL responding cells are PRL containing and secreting, we cannot say at present whether PRL also acts in a paracrine fashion on GH secretors. The results with added purified PRL are interesting in that they raise the possibility that another one of the isoforms of 24-kD PRL is inhibitory to proliferation. Unlike GH3 cells, normal cells in culture secrete isoforms 3 and 3’ in addition to isoform 2 (16). Isoforms 3 and 3’ are formed from isoform 2 by posttranslational phosphorylation in secretory granules (16), secretory granules that are usually essentially absent from GHJ cells (17). The added PRL was a mixture of isoforms containing a substantial proportion of isoforms 3 and 3’. It was added in amounts sufficient for isoforms 3 and 3’ to be significant vs. the isoform 2 produced by the cells. These amounts are also representative of hypothalamohypophyseal portal concentrations. Given the amount of PRL secreted by the GH3 cells, which is all isoform 2, and the proportion of isoform 2 in the added PRL, this result further suggests that isoform 3 and/or 3’ must be a more potent inhibitor than isoform 2 is a stimulator of proliferation. It is also interesting to note that at high densities, where we can FIG. 7. Colocalization of cell surface PRL (I, left) and PRL teceptor (II, right). Split images from confocal screen. This time the receptor was visualized with a Texas red-conjugated second antibody and PRL with a fluorescein-conjugated second antibody. In A note the similar regional distribution of PRL and PRL receptors. Intense Texas red fluorescence tended to obscure the more punctate localization of the receptors. B shows the receptor control where Ug was replaced by OKT4. Note the punctate distribution of surface PRL (I) and absence of receptor fluorescence (II). C shows the surface PRL control where IC4 was replaced by normal rabbit serum. No PRL fluorescence is seen on left side. In this instance the receptors (right side) appeared to show a more homogeneous cell surface distribution. Bar in A, 3 pM; bars in B and C, 10 pM.


PRL AS AN AUTOCRINE

GROWTH

FACTOR

5. Cuttitta F, Carney DN, Mulshine J, Moody TW, Fedorko J, Fischler A, Minna JD 1985 Bombesin-like peptides can function as autocrine growth 316:823-826

factors

in human

6. Brelje TC, Sorenson RL 1991 hormone on islet B-cell proliferation nancy. Endocrinology 128:45-57

small-cell Role

lung

cancer.

Nature

of prolactin versus growth in vitro: implications for preg-

7. Yang J, Richards J, Guzman R, Imagawa W, Nandi S 1980 Sustained growth cells embedded

2092 8. Hartmann phocyte

in primary in collagen

culture of normal mammary epithelial gels. Proc Nat1 Acad Sci USA 77:2088-

DP, Holaday JW, Bernton EW 1989 Inhibition

proliferation

by antibodies

to prolactin.

FASEB

of lymJ 3:2194-

2202 9. Green LM, Reade JL, Ware CF 1984 Rapid colormetric 10.

11.

12.

FIG. 8. Cell surface was found

and intracellular PRL. Note that on PRL-containing cells (bar, 10 pm).

cell surface

PRL 13.

be sure that PRL-driven proliferation is maximal, added PRL resulted in the net loss of the same number of cells (-200,000) at each density. This suggeststhat binding to a given number of precursor cellscompletely inhibited division of those cells. Dephosphorylation of the added NIDDK PRL proved that the inhibition was indeed due to the phosphorylated isoforms. Using the same experimental approaches, we have also been able to demonstrate a PRL autoreceptor on normal mammotrophs (data not shown). Although we hesitate to extend results with a cell line to interpretations concerning normal pituitary cells, it is nevertheless interesting to speculate that changes in the secreted isoform profile toward a higher percentageof isoform 2 may result in the proliferation of normal mammotrophs. Such a change might be predicted when proliferation is stimulated under physiological circumstances such as pregnancy and under pathological circumstancessuch as prolactinoma. Indeed we have documented increased ratios of isoforms 2:3 in pregnancy (35, 36) and associatedwith proliferation during prolactinoma development (35). In summary, we have demonstrated that PRL isoform 2 is a growth factor for a subsetof GH3 cells and have produced preliminary evidence suggesting that one of the other isoforms of 24-kD PRL may be inhibitory to proliferation.

Costello RT 1936 Subclinical

adenoma

16.

2048 17. Tixier-Vidal 18. 19. 20.

21.

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gland.

Am

3. Dembinski 4.

for the recurrent

TC, Shiu RPC 1987 Growth development and function. In Neville Mammary Gland. Plenum Press, New Smith JJ, Derynck R, Korc M 1987 growth factor 01 in human pancreatic superagonist autocrine cycle. Proc Nat1

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adenoma.

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factors in mammary gland MC, Daniel CW (eds) The York, pp 355-381 Production of transforming cancer cells: evidence for a Acad Sci USA 84:7567-7570

A 1975 Ultrastructure of anterior pituitary cells in culture. In: Farquhar MG, Tixier-Vidal A (eds) The Anterior Pituitary. Academic Press, New York, pp 181-229 Haug E, Tjernshaugen H, Gautvik KM 1976 Variations in prolactin and growth hormone production during cellular growth in clonal strains of rat pituitary cells. J Cell Physiol 91:15-30 Melmed S, Carlson HE, Briggs J, Hershman JM 1980 Autofeedback of prolactin in cultured prolactin-secreting pituitary cells. Horm Res 12:340-344 Danielpour D, Ikeda T, Kunkel MW, Sirbasku DA 1984 Identification of an autostimulatory growth factor in the extracts and conditioned medium of GH3/C14 rat pituitary cells in culture. Endocrinology 115:1221-1223 Hinkle PM, Kinsella PA 1986 Thyroid hormone induction of an autocrine growth factor secreted by pituitary tumor cells. Science

234:1549-1552 22. Sirbasku DA, Pakala R, Sato H, Eby JE 1991 Thyroid

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12:205-216 Clark K 1980 Surgery Neurosurg 27:309-314

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Prolactin synthesized and secreted by human peripheral blood mononuclear cells: an autocrine growth factor for lymphoproliferation.

Inhibition of prolactin gene transcription by transforming growth factor-beta in GH3 cells.

Identification of interleukin-6 as an autocrine growth factor for Epstein-Barr virus-immortalized B cells.

A 41 kDa transferrin related molecule acts as an autocrine growth factor for HL-60 cells.

FGF19 functions as autocrine growth factor for hepatoblastoma.

Epstein-Barr virus-immortalized B cells produce IL-6 as an autocrine growth factor.

Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells.

Effects of resveratrol on cell growth and prolactin synthesis in GH3 cells.

Low-molecular weight B cell growth factor (BCGF-12KD) as an autocrine growth factor in B cell chronic lymphocytic leukemia.

paracrine promoters of progression of hepatocellular carcinoma.

Parathyroid hormone related peptide can function as an autocrine growth factor in human renal cell carcinoma.

Basic fibroblast growth factor: an autocrine mitogen of rat thyroid follicular cells?

paracrine roles of extrapituitary growth hormone and prolactin in health and disease: An overview.

Autocrine growth of leukemic cells.

paracrine regulation of prolactin release from human decidual cells.

Endothelin as an autocrine factor in the regulation of parathyroid cells.

Epidermal growth factor dependence and TGF alpha autocrine growth regulation in primary rat tracheal epithelial cells.

Fibroblast growth factor receptor 4 (FGFR4) and fibroblast growth factor 19 (FGF19) autocrine enhance breast cancer cells survival.

paracrine factor for human tumor cell lines.

Identification of an autocrine negative growth factor: mouse beta-galactoside-binding protein is a cytostatic factor and cell growth regulator.

Effect of the GABAA agonist muscimol on prolactin secretion from human prolactin-secreting adenomas and GH3 rat pituitary tumour cells.

Secretion of insulin-like growth factor II (IGF-II) and IGF-binding protein-2 by intestinal epithelial (IEC-6) cells: implications for autocrine growth regulation.

Vasoactive intestinal peptide: autocrine growth factor in neuroblastoma.

Potentiation of thyrotropin-releasing hormone-stimulated prolactin mRNA levels in GH3 cells by acetylcholine.