Molecular and Cellular Endocrinology, 18 ( 1980) 24 l-248 0 Elsevier/North-Holland Scientific Publishers, Ltd.

241

DNA POLYMERASES IN THE RAT PITUITARY GLAND

EFFECT OF OESTROGENS AND SULPIRIDE

Graciela A. JAHN, Liliana E. KAL~~~ANN, Gloria MACHIAVELLI, Irena SZIJAN and Jose A. BURDMAN Departamento de Q&mica Biol&ka, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Buenos Aires (Argentina)

Received 14 September 1979;accepted 26 February 1980

Changes in the activity of DNA polymerase and [ 3H] thymidine incorporation into the DNA of the anterior pituitary gland were studied in oestrogenized male and pregnant rats.

The activities of DNA polymerases 01 and p, extracted in Tris-HCl or in sodium phosphate buffer were characterized according to their optimum pH and sensitivity to N-ethyl-maleimide. In the Tris-soluble fraction DNA polymerase activity is almost exclusively LY, while in the phos- phate soluble fraction it is a mixture of a: and p.

The administration of oestrogens to male rats increases 13H]thymidine incorporation and enhances the activity of DNA polymerase in the Tris-soluble fraction, while the activity of the phosphate-soluble enzyme does not change. Sulpiride administration results in a further incre- ment of [ 3H] thymidine incorporation and of DNA polymerase activity in the Tris-soluble frac- tion.

In pregnant rats sulpiride also produces an increment of DNA polymerase activity only in the Tris-soluble fraction.

Thus, the activity of the Tris-soluble fraction from APG behaves as DNA polymerase 01. This activity changes in parallel with [ 3H] thymidine incorporation into DNA which is an indication of cell proliferation in the gland.

This is discussed with respect to a negative feedback mechanism between intracellular pro- lactin concentration and DNA synthesis in the APG.

Keywords: anterior pituitary gland; prolactin release; oestrogens; DNA polymerases; pro- liferation control.

In previous work we have studied the incorporation of [3H]thymidine in the anterior pituitary gland (APG) during development, pregnancy and after the admin- istration of drugs affecting the release of prolactin from the gland (Kalbermann et al., 1979a,b). The incorporation of the DNA precursor in the APG diminishes during development (Kalbermann et al., 1979a; Mastro et al., 1969) and is lower in rats in late pregnancy than in virgins (Kalbermann et al., 1979b). Sulpiride, a drug known to produce prolactin release from the APG (Debeljuk et al., 1975) increases

242 Graciela A. Jahn et al.

the incorporation of [3H]thymidine into the DNA of the gland in pregnant and male rats (Kalbermann et al., 1979a,b). This rise is significantly decreased when bromoergocryptine, which blocks prolactin release from the APG, is administered along with sulpiride (Kalbermann et al., 1979b). Clomiphene, an oestrogen recep- tor-blocking agent, completely abolishes the rise in [3H]thymidine incorporation produced by sulpiride. On the other hand, clomiphene does not affect the prolactin release produced by sulpiride (Burdman et al., in press). These results suggest that the liberation of prolactin induces a proliferative response in the APG, and that this response is mediated by oestrogens.

We have also shown that changes in the activity of Tris-soluble DNA polymerase (deoxynucleoside triphosphate: DNA deoxynucleotidyl transferase, EC 2.7.7.7) are parallel with those of [3H]thymidine incorporation during development and preg- nancy (Kalbermann et al., 1979a,b). On the contrary, a phosphate-soluble DNA polymerase (Kalbermann et al., 1979a), as well as thymidine kinase and endonu- clease activities do not change significantly (Kalbermann et al., 1979b). It seems logical to postulate an important role of the Tris-soluble polymerase activity in the variations of [3H] thymidine incorporation mentioned above.

The purpose of this work is to study the relationship between the changes pro- duced by sulpiride in the Tris- and phosphate-soluble activities of the DNA poly- merases and [3H]thymidine incorporation. For this purpose we studied the effect of sulpiride in oestrogen-treated males and pregnant rats.

Of the 3 main DNA polymerases that occur in mammalian cells, the most studied, and most easily distinguishable are DNA polymerases CY and /3 (Weissbach, 1977). We tried to characterize the Tris- and phosphate-soluble DNA polymerases as (Y or /3 enzymes, or a mixture of both, taking advantage of their different pH optima and sensitivity to N-ethyl-maleimide (NEM), an SH-group blocking agent

(Weissbach, 1977). We found that the changes observed in [3H]thymidine incorporation are paral-

lel to the variation in the Tris-soluble DNA polymerase, and that this activity is

mainly composed of the 01 enzyme.

MATERIALS AND METHODS

Animals. Rats from a highly inbred Wistar strain were used. They were kept in conditions of 12 h light, 12 h darkness with food ad libitum. Male rats weighed 200-250 g. Pregnant rats were killed l-4 days before delivery.

Treatments. 5 mg/rat of sulpiride sulphate (Vipral, Roemmers, Argentina) in 0.5 ml of 0.14 M NaCl was injected subcutaneously 20 h before decapitation of animals. Controls received the same volume of the vehicle.

50 ,ug/rat of 3,17-oestradiol diundicelinate (Etrosteron, Gador, Argentina) dis- solved in 0.2 ml oil solution were injected subcutaneously, daily during 6 days. On

DNA polymerases in pituitary gland 243

the 6th day the rats were injected with 5 mg of sulpiride sulphate, immediately after the oestrogen injection and decapitated 20 h after. Controls received the same volume of the vehicles.

Incorporation of [3H/thymidine. The animals were decapitated, the pituitary glands were removed, the posterior lobe discarded, the anterior pituitary glands halved and transferred to chilled tubes containing 0.5 ml of medium TC 199 (Mor- gan et al., 1950) and incubated under 95% 0*-S% CO2 at 37°C in a metabolic shaker. After 10 min 2 /&i of [Me-3H] thymidine (spec. act. 50.8 Ci/mmole) were added and the incubation continued for another 60 min. During this time the incorporation of the precursor was linear. At the end of incubation, the medium was rapidly removed and the APGs were washed twice with 1 ml of cold 0.14 M NaCl. The tissue was then homogenized in 1 ml 10% (w/v) cold trichloroacetic acid solution (TCA) and centrifuged at 6500 X g for 15 min. The insoluble residue was washed twice with 1 ml of 5% (w/v) TCA solution and dissolved in 0.3 ml of 0.5 N NaOH. Aliquots of this solution were taken to determine: (a) DNA by the diphenylamine reaction with native DNA as a standard (Burton, 1956) and (b) radioactivity in vials containing 10 ml of Bray’s solution and 500 mg of Cab-O-S1 Ms (Cabot, Argentina). The supernatants were used to measure the radioactivity in the soluble fractions and represent the precursor uptake by the tissue. Counts were

corrected to 100% efficiency by the channel ratio method.

Preparation of extracts for DNA polymerase. The glands were rapidly removed, weighed (between 35-45 mg) and homogenized in 1 ml 10 mM Tris buffer (pH 7.4), 2 mM /3-mercaptoethanol and centrifuged 20 min at 24 150 X g. The superna- tant (Tris-soluble fraction) was used to determine enzymatic activities and protein content. In some cases the pellet was extracted with 1 ml of 0.2 M phosphate buffer pH 7.4, 2 mM /3-mercaptoethanol, allowed to stand at 0°C during 30 min and centrifuged 20 min at 35 600 X g. The supernatant (phosphate-soluble fraction) was used to determine protein content and DNA polymerase activity.

DNA polymerase assay. This assay measures the incorporation of [3H]dTMP into an acid-insoluble product. The incubation mixture contained 20 pmoles of Tris buffer (pH 7.2 or 8.6), 2 pmoles of MgC12, 2 pmoles of pmercaptoethanol, 20 nmoles each of dATP, dGTP and dCTP, 500 pmoles of dTTP, 300 nmoles of ATP, 2 .&i of [3H]dTTP (63.2 Ci/nmol), 25 pg of activated DNA and an appropriate

amount of the enzyme preparation, 180-220 pg protein approximately for the Tris-soluble fraction and loo-125 pg protein approximately for the phosphate- soluble fraction in a final volume of 0.4 ml. The reaction mixture was incubated at 37°C for 15 and 30 min. The reaction was stopped and the radioactivity mea- sured as described previously (Szijan and Burdman, 1974).

Activated DNA was prepared as described previously (Kalbermann et al., 1979a). For the assays measuring NEM inhibition, this drug was added to the reaction

244 Graciela A. Jahn et al.

mixture to give a final concentration of 10 mM. The tubes were kept for 20 min at 0°C before incubating.

Chemical determination of DNA and proteins. DNA was measured according to the method of Burton (Burton, 1956). Protein was determined by the method of

Lowry (Lowry et al., 1951).

Statistical analysis was performed using Student “t” test.

RESULTS

It is well known that the activities of DNA polymerases o. and J!I can be differ- entiated using specific inhibitor (NEM) and different pHs (Weissbach, 1977).

Table 1 shows that the enzymatic activity in the Tris-soluble fraction from the APG behaves like the Q! enzyme, its activity is almost 3 times higher at pH 7.2 than at pH 8.6, and is almost completely inhibited by NEM 10 mM. This indicates that the DNA polymerase activity present in this fraction is mainly the (Y enzyme. The enzymatic activity in the phosphate-soluble fraction, on the contrary, behaves like a mixture of (Y and 0 DNA polymerases, it shows higher activity at pH 8.6 but is partially inhibited by NEM 10 mM. We can assume that the values obtained for the phosphate-soluble fraction in the assay with NEM 10 mM at pH 8.6 represent the activity of a P-DNA polymerase. We have not found DNA polymerase activity in the residue after successive extraction with both solutions.

All the assays were performed in duplicate, measuring the activity at 15 min and 30 min incubation time. Fig. 1 was obtained using all the DNA polymerase determinations of this work. The values of the 30 min determinations were taken as

Table 1

Tris- and phosphate-soluble DNA polymerase activities from the APG of male rats Effect of pH and NEM.

pH 7.2

-NEM

10 mM

+NEM

10 mM

pH 8.6

-NEM 10 mM

+NEM

10 mM

Tris-soluble enzyme

Phosphate-soluble enzyme

45.0 f 5.3 4.0 f 0.6 185 f 3.5 3.0 * 3.0

(9%) (16%) 15.0 f 3.0 10.0 f 3.0 33.0 f 2.1 17.0 f 1.2

(67%) (52%)

DNA polymerase assay was performed as described in Materials and Methods. Data are

expressed as pmol dTMP incorporated/mg protein/30 min of incubation * S.E.M. The percent- age represents the remaining activity.

DNA polymerases in pituitary gland 245

Time (min)

Fig. 1. Linearity of the DNA polymerase assay. All the DNA polymerase determinations done

in this work were used. The values of the 30 min determinations were taken as 100%. The

15 min point is an average of each 15 min assay expressed as a percentage of the corresponding

30 min determination *S.D. The average value of the 15 min point is 55.1 ? 5.0%.

1 OO%, the 15 min point is an average of the values of each 15 min assay expressed as a percentage of the corresponding 30 min determination. It can be seen from Fig. 1 that the reaction was linear up to 30 min in all cases.

We used oestrogen-treated males and pregnant rats for the following experiments because the increase in [3H]thymidine incorporation in response to sulpiride is much greater in these animals than in virgin rats or untreated males.

Table 2 shows the effect of oestrogens and oestrogens plus sulpiride in the [3H]- thymidine incorporation into the DNA of the APG of male rats. Oestrogen alone produces an increase in the [3H]thymidine incorporation. However, when sulpiride

is administered to an oestrogenized rat the increase is strikingly more pronounced. Table 3 shows that oestrogen treatment increases the activity of the DNA poly-

Table 2

“In vitro” incorporation of [ 3H] thymidine into DNA of male rat APG Effect of oestrogens and sulpiride.

Treatment Specific relative

radioactivity f S.E.M. % Change

Vehicle

Oestrogens Oestrogens + sulpiride

91 f 10 -

206 f 15 a) 109 519+ 107a) 435

Relative specific radioactivity = insoluble dpm/mg DNA x 1ooo

’ soluble dpm/mg DNA a) P < 0.01 between this value and vehicle.

Results are the average of 3 Expts.

246 Graciela A. Jahn et al.

Table 3

DNA polymerase activities in male rat pituitary gland

Effect of oestrogens and sulpiride.

Treatment Tris-soluble fraction Phosphate-soluble fraction

-NEM +NEM -NEM +NEM

10mM 10 mM 10mM 10 mM

Vehicle

Oestrogen

45.0 * 5.3 4.0 + 0.6 33.0 f 2.1 17.0 * 1.2

89.0 f 2.6 a) 7.0 zk 0.7 c) 38.0 * 3.6 b) lg.o+ 1.4 b)

(97%) Oestrogen + sulpiride 136.0 f 10.4 a) 11.0 r 2.1 c) 42.0 f 2.0 C) 16.0 + 1.4 b)

(202%)

Results are the average of 5 Expts. Data are expressed as pmoles TMP incorporated/mg protein/

30 min of incubation f S.E.M. DNA polymerase assay was performed as indicated in Materials

and Methods. The Tris-soluble fraction was assayed at pH 7.2 and the phosphate-soluble frac-

tion at pH 8.6. a)J’ < 0.0005 between this value and the corresponding value of vehicle.

b) n.s. e) P < 0.05 between this value and the corresponding value of vehicle.

merase (IL present in the Tris-soluble fraction, and the administration of sulpiride ‘increases even further the activity of this enzyme. On the other hand, DNA poly- merase /3, represented by the activity of the phosphate-soluble fraction in the pres- ence of NEM 10 mM does not change significantly.

Table 4 shows that the DNA polymerase sensitive to NEM 10 mM (CY enzyme) in the Tris-soluble fraction also rises markedly when pregnant rats are treated with sulpiride. The activity in the phosphate-soluble fraction, insensitive to NEM inhib- ition, again does not change (data not shown).

Table 4

Tris-soluble DNA polymerase activity in pregnant rats

Effect of sulpiride.

Treatment -NEM 10 mM +NEM 10 mM

Vehicle Sulpiride

38.0 f 1.3 6.0 + 3.0 65.0 + 7.3 a) (72%) 7.0 + 1.6 b)

Data are expressed as pmoles TMP incorporated/mg protein/30 mm of incubation k S.E.M. Results are the average of 5 Expts. DNA polymerase assay was performed as indicated in Mate-

rials and Methods. The assay was done at pH 7.2. a) P < 0.005 between this value and vehicle.

b) n.s.

DNA polymeraxes in pituitary gland 241

DISCUSSION

Cell proliferation in the APG varies with the physiological state of the animal, for example during the oestrous cycle (Hunt and Hunt, 1966) during development (Kalbermann et al., 1979a; Mastro et al., 1969) pregnancy (Kalbermann et al., 1979b) and lactation (Hunt, 1949). It can also be stimulated to proliferate in

response to hormonal and therapeutic agents (Kalbermann et al., 1979a,b; Lloyd et al., 1978). [3H]Thymidine incorporation into the DNA of the APG correlates closely with the mitotic index and hence is a good measure of cell proliferation (Hunt, 1949). Our previous results demonstrate a decrease in [3H]thymidine incor- poration and in the activity of a Tris-soluble DNA polymerase during development in the APG (Kalbermann et al., 1979a). On the other hand, Mastro and Hymer (1973) have shown that [3H]thymidine incorporation and the activity of a cyto- plasmic DNA polymerase in the APG of male rats diminish during development and rise after oestrogen treatment. To our knowledge the activities of DNA polymerases QI and /3 in the APG were not characterized up to now. In this work we have been able to demonstrate that DNA polymerase 01 activity increases when the gland is stimulated to proliferate. In other tissues the activity of DNA polymerase 01 is high when active proliferation is taking place (Baril et al., 1973; Roodinan et al., 1976) and during the S phase of the cell cycle (Spadari and Weissbach, 1974). On the other hand, the activity of DNA polymerase 0, found mainly in the phosphate soluble fraction from the APG does not change when this tissue is stimulated to proliferate. Again this is in agreement with the findings in other tissues (Roodinan et al., 1976; Spadari and Weissbach, 1974).

We have previously shown that sulpiride treatment produces a small but signifi- cant increase in thymidine incorporation and in the Tris-soluble DNA polymerase in male rats (Kalbermann et al., 1979a). However, when sulpiride is administered to an oestrogenized rat these rises are much more pronounced. Oestrogen treatment alone also increases these activities, but the rise produced by sulpiride administration to an oestrogenized rat is higher than the sum of the effects produced separately by both treatments.

Sulpiride treatment in pregnant rats also produces a marked rise in [3H]thym- idine incorporation into the DNA of the APG (Kalbermann et al., 1979b). In this situation the changes in the activity of the DNA polymerase 01 are also parallel to the changes in the incorporation of the DNA precursor.

Lloyd et al. (1975) and ourselves have proposed that the acute transitory deple- tion of prolactin triggers a series of events which result in an increase in DNA synthesis in the APG. Moreover the rise in DNA synthesis could be due, at least in part to the rise in activity of a DNA polymerase that behaves like the o-enzyme.

248

ACKNOWLEDGMENTS

Graciela A. Jahn et al.

We are grateful to Prof. Carlos J. G6mez for the opportunity to perform this work. These studies were supported by grants from the Consejo National de Inves- tigaciones Cientificas y TCcnicas, from the Comisi6n National de Energia Atbmica (Argentina) and by PLAMIRH (99.178.1.78).

REFERENCES

Baril, E.F., Jenkins, M.D., Brown, O.E., Laszlo, J., and Morris, H.P. (1973) Cancer Res. 33,

1187.

Burdman, J.A., Szijan, I., Jahn, G.A., Machiavelli, G., and Kalbermann, L.E., Experientia, in

press.

Burton, K. (1956) Biochem. J. 62, 315.

Debeljuk, L., Rozados, R., Daskal, H., Villegas-Velez, C., and Mancini, A.H. (1975) Proc. Sot.

Exp. Biol. Med. 148, 550.

Hunt, T.E. (1949)Anat. Rec. 105, 361.

Hunt, T.E., and Hunt, E.A. (1966) Anat. Rec. 186, 361.

Kalbermann, L.E., Szijan, I., Jahn, G.A., Krawiec, L., and Burdman, J.A. (1979a) Neuroendo-

crinology 29, 42.

Kalbermann, L.E., Szijan, I., and Burdman, J.A. (1979b) Experientia 35, 689.

Lloyd, H.M., Meares, J.D., and Jacobi, J.M. (1975) Nature (London) 255, 497.

Lloyd, H.M., Jacobi, J.M., and Meares, J.D. (1978) J. Endocrinol. 77, 129. Lowry, O.H., Rosebrough, H.J., Farr, A.L., and Randall, R.J. (1951) J. BioL Chem. 193, 265.

Mastro, A., and Hymer, W.C. (1973). J. Endocrinol., 59, 107.

Mastro, A., Hymer, W.C., and Therrieu, CD. (1969) Exp. Cell Res. 54, 407.

Morgan, J.F., Morton, H.J., and Parker, R.C. (1950) Proc. Sot. Exp. Biol. Med. 73, 1.

Roodinan, G.D., Hutton, J.J., and Bollum, F.J. (1976) Biochim. Biophys. Acta 425, 478.

Spadari, S., and Weissbach, A. (1974) J. Mol. Biol. 86, 11.

Szijan, I., and Burdman, J.A. (1974) Brain Res., 73,563.

Weissbach, A. (1977) Annu. Rev. Biochem. 46, 25.