Hydrocortisone-dependent alterations of transcriptional activity in WI38 cells

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Cell Research 122 (1979) 179-184

HYDROCORTISONE-DEPENDENT TRANSCRIPTIONAL J. M. RY.4N’

ACTIVITY

ALTERATIONS

OF

IN WI38 CELLS

and V. J. CRISTGF.\LO

SUMMARY The sffect of hydrocortisone on the transcriptional activity of exponentially and parasynchronously growing WI38 cells was measured using E. coli RNA polymerase or endogenous chromatinbound RNA polymeras to catalyse RNA synthesis in vitro. Results indicated that the transcriptional activity of cells was increased 8 h after hydrocortisone addition to exponentially growing cultures and 3-5 h after hormone addition to parasynchronously growing cultures. The hydrocortisone-induced alteration of transcriptional activity was not an artifxt induced by different rates of RN.4 or DNA degradation. These findings indicate that the hormone-dependent alteration of transcriptional activity is an early event in the hydrocortisone-mediated stimulation of WI38 cell proliferation.

The growth rate and lifespan of the human hydrocortisone. The results of these experidipioid cell lines WI38 and IMR-90 can be ments form the basis of this report. modulated by addition of hydrocortisone to the growth medium [l-3]. Typically, exMATERIALS ,4ND METHODS posure of cell cultures to 14 PM hydrocortisane results in an extended period of expo- Cell culture nential growth and a 20-3075 increase in The human diploid cell line WI38 was cultivated in the of antibiotics as previously described [7, 8:. cell number at stationary phase. Lifespan absence Only cultures between the 29th and 39th population increases of 30-405X occur when cells are doubling level (>SO”c labeled nuclei) [7] were used in this study. This was done to minimize the influence of serially propagated in the presence of the age-associated functional alterations [9] on the cellular hormone [2]. response to hydrocortisone. Cells were inoculated at 0.5-1.0~ IO4 cells/cm’ and The mechanism by which hydrocortisone reached confluency 5-7 days later. Parasynchronocsiy promotes cell division is not known. How- growing cultures were obtained by refeeding cultures Z-3 davs oost-conflurncv with fresh medium conever, glucocorticoids have been reported to taining !55%serum with and without 14 ,&I hydrocorrialter the transcriptional activity of tissue sone. The length of the ore-DNA svnthetic period tof cek was determined by pulsing cultures and organ cultures [4&j, which suggests stimulated with 0.1 pCi/rnl [3H]thymidine ([“H]TdR) for 0.5 h that gene activation or amplification is hor- every other hour after stimulation. Cellular DNA was pr2cipitated with cold IO% trichloroacetic acid (TC,L\). mone dependent. collected on Whatman GF/c filters. washed with IOr5 To better understand the action of hydro- TCA and 95 % ethanol, and digested with 1 ml Protosol (New England Nuclear, Boston, Mass.). The amount cortisone at the molecular level, we have of r3H]TdR incorporated into DNA was determined b! measured the transcriptional activity of exponentially and parasynchronously growing L Present address: IIT Research Institute. 10 West 35 WI38 cell cultures after the addition of Street. Chicago, IL 60616. USA.

180

Ryan and Cristofalo

Table 1. Transcriptional acti\lity of chrornatin isolated from control and hydracortisone-treated cells Transcriptional activity (cpmlpg DNA) Time exposed to hydrocortisone (hours) 1

6 7 Average t S.E. ; 9 9 72 72 ;2verage f S.E.

Control

Hormonetreated

I 448 I 838 ? 578 i 955-t331

I417 I 461 2 541 1 807+367

21 929 578 I 932 I 752 1 281 1 582 1 842k178

32 232 244 1 198 :! 611 1 690 2 061 2 339t?l6

Hydrocortisone (14 FM) was added to exponentially growing cultures for the times indicated. Chromatin from untreated and hormone-treated cells was isolated and assayed for transcriptional activity by using an excess of E. co/i RN.4 polymerase. Transcriptional activity of chromatin from hydrocortisone-treated cells differs significantly from control values (28 h; p< 0.005) when evaluated using the paired Student’s Itest.

RN.4 was collected, washed and prepared for scintillation counting as described above.

Measurement of degradative acti\+t) of isolated nuclei Y

RNAse activity of isolated nuclei was measured by incubating 59 pF(g[3H]RN4 (7000 cpm/pg RNA) with nuclei (13 yg DNA) isolated (by method B) from exponentially growing hydrocortisone-treated (24 h) and control cells. After 60 min incubation at 37°C. the labeled RN.4 was precipitated with cold IO% TCA. and the amount of radioactivity present was determined as described earlier. Degradation of the DNA template of isolated nuclei from control and hydrocortisone-treated (24 h) cells was determined bv incubating chromatin (prepared by method Aj containing 9 pi DNA prelabeied with r3H1TdR t7 000 cDm/ug DNA) at 37 “C for 60 min and Lo&paring the amo&t of piecipitable [“H]DNA of incubated and non-incubated samples. All determinations of DN?rse or RNAse activities were made in the same medium used to measure RNA synthesis. In all experiments, 1 mg of carrier RNA or DNA was added before precipitation of RNA or DNA. [3HJRN.4 was prepared from exponentially growing cells using the method described by Rovera et al. [13]. [3H]DNA was prepared by pulsing exponentially growing control and hormone-treated cells with 0.1 Ki/ml T3H1TdRfor 24 h and then isolating chromatin king method B. The radiochemicals [“H]TdR (spec. act. 2.Ciimmolej and [3H]YTP (spec. act. 28 Ci/mmole) were obtained from New England Nuclear, Boston, Mass.

counting the radioactivity in a SL-30 Intertechnique liquid scintillation spectrometer.

Measurement of transcriptional activiry The transcriptional activity of parasynchronously or exponentially growing untreated (control) or hydrocortisone-treated cells was determined by one of two different methods. In method A, chromatin was isolated from hydrocortisone-treated and control cells and transcriptional activity was determined as described by Augenlicht & Baserga [lo]. The chromatin (9 pg DNA) was incubated with a non-rate limiting concentration of E. cob RNA oolvmerase fraction IV (16 units) (GIBCO, Grand Island,*NY). In method B, nuclei from control and hormone-treated cells were isolated, and the rate of RNA synthesis was measured using only the chromatin-bound RNA polymerase to catalyse RNA synthesis [11, 121. In both methods, RNA svnthesis was conducted in the presence of O.u.5 -PM adenosine triphosphate (AT@), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and 10 &i [jJH]uridine triphosphate ([3H]UTP). RNA synthesiswascarriedoutat37 “Cfor lOmininmethod.4 and at 23 “C for 20, 40 and 60 min in method B. After the appropriate incubation period, 1 mg RNA was added to each sample, and transcription was terminated by addition of cold 5 ‘% TCA. The precipitated E.xp Cell Res I22 (1975)

RESULTS Hydrocortisone-induced alterations in transcriptional acti\lity in exponentially growing Cells To determine whether hydrocortisone altered the rate of transcriptional activity of WI38 cells, 14 PM hydrocortisone was added to exponentially growing cells. The chromatin from untreated and hormone-

Table 2. RNAse activities of isolated nuclei Nuclei from cells

cpm/pgDNA

Unstimulated FM FM+HC [3H]RNA without nuclei [3H]RNA+ 1 mg RNAse

7 003 7 019 6 907 7 017 0

Altemtions yftrnnsci’iptionnl

fig. 1. .4bscissa: time after stimulation of confluent ceils ihours): ordinare: total corn x IO-’ PHlTdR incorpoiated into DNA of unstiklated c&Is -(G--O): cells stimulated with fresh medium with (0-O); or without hydrocortisone (A-A). Transcriotional activitv (cumiurr DNA) of isolated chromatin ‘from unstimuia;eh 0; ?resh medium with (FM+HC) and without (FM) 14 uM hvdrocortisone was measured every 5 h posi-stimilation (using method A). Values obtained (cpm/Fg DNA) are shown in the table inset.

nctil,iry

181

porated into RNAIpg DNA for control and hydrocortisone-treated cells was 18425 178 and 2 339+2 16 cpm/pg DNA, respectively (average of six determinations). and was significant at thep
H~cl~ocor-tisone-deper?dentaltemtionz in par-cts~nci~r-o~~or~s~~ gro,tGg cell5 To determine if hormone-dependent alterations in transcriptional activity could be detected earlier than 8 h after addition of hydrocortisone and to ascertain whether the increase in transcriptional activity was treated cells was isolated. and the transcrip- associated with a specific phase of the ceil tional activity was determined. Table 1 cycle. confluent ceils (arrested in GO-&l, shows the results obtained by using method were stimulated to undergo DNA synthesis A to determine transcriptional activity. by addition of fresh medium with FM+HCj During the first 7 h after the addition of or without (FM) hydrocortisone. Subsehydrocortisone, there was no difference in quent changes in transcriptional activity the transcriptional activity of hormone- were correlated with alterations in the rate treated as compared with non-treated cells; of [3H]TdR incorporated into the DNA of the average specific activity +S.E.M. of stimulated cultures (fig. 1). The results intreated ceils was 1807+367 cpm/pg DNA dicated that the pre-DNA synthesis period and that of untreated cells was 1955+331 of FM- and FM+HC-stimulated cultures cpm/pg DNA. However, the transcrip- was about 12 h, and the period of active intional activity of chromatin isolated from corporation of [3H]TdR into DNA occurred cells treated with hydrocortisone for 8 h 12-24 h post-stimulation. There was essenand longer was significantly increased over tially no difference in the time required for that of untreated cells. The average cpm of FM+ HC- and FM-stimulated cell populauridine monophosphate ([3H]UMP) incor- tions to initiate DNA synthesis, or to com-

182

Ryan and Cristofalo activity occurred earlier than 5 h poststimulation of confluent cells with FM+HC (using method B) indicated that a consistent increase in transcriptional activity could be resolved as early as 3 h after addition of fresh medium containing hydrocortisone to confluent cells (fig. 2). Results 1 and 2 h post-stimulation varied, however (data not shown).

20 16 16 14 12 IO 6 6 4 2 I 20

40

60

Fig. 2. Abscissa: time of incubation of isolated nuclei (min); ordinate: [3H]UMP incorporated into RNA cpm x 10-3/pg DNA. Transcriptional activity (method B) of nuclei from unstimulated cells (O), cells stimulated 3 h with fresh medium (a) or fresh medium containing 14 PM hydrocortisone (D).

plete DNA synthesis. Unstimulated cell cultures maintained a low, relatively constant rate of [3H]TdR incorporation into DNA throughout the experimental period. When the transcriptional activity of unstimulated, FM- and FM+HC-stimulated cultures was compared every 5 h (table inset, fig. 1) during the 25-h period (using method A), it was found that both FM- and FM+HC-treated cultures responded with increased transcriptional activity within 5 h after medium renewal. However, FM+HC cells had an increased activity over FMtreated cells (3 992 vs 3 190 cpm/pg DNA). As the cell population of both cultures approached (10 h) and progressed through the S phase of the cell cycle at 15 and 20 h poststimulation, the transcriptional activity of both FM- and FMSHC-stimulated cells declined to control values, but increased slightly in both cultures as an increased number of cells completed DNA synthesis (25 h post-stimulation). Studies to determine whether hydrocortisone-dependent changes in transcriptional Exp Cell Res 122 (1979)

RNAse and DNAse activity of isolated chromatin Rather than increasing the transcriptional activity of treated cells, hydrocortisone could stabilize or protect the isolated chromatin from RNAse or DNAse digestion. When the RNAse activity of nuclear preparations of exponentially growing control and hydrocortisone-treated (24 h) cells were compared (table 2), there was no difference. However, when the r3H]RNA was incubated with these same nuclear preparations plus RNAse, the [“H]RNA was digestible. To determine the DNAse activity of isolated nuclei, nuclei (9 pg DNA) pre-labeled with [3H]TdR were isolated from control and hydrocortisone-treated cells (24 h) by method B, and the amount of precipitable r3H]DNA was determined after 60 min incubation at 37 “C. The results indicated that 76k 11% and 80+6% of the [3H]DNA of control and hydrocortisone-treated cells, respectively, was precipitated. DISCUSSION Although the fidelity of bacterial RNA polymerase directed transcription of isolated chromatin is unclear, these results combined with the hormone-dependent increase in transcriptional activity using isolated. nuclei indicate that hydrocortisone acts to increase the chromatin template activity of WI38 cells. Hormone-dependent al-

terations of transcriptional activity have been described in other tissue and organ cultures [l&17]: however. the hydrocoitisone-mediated increase in transcriptional activity of WI38 cells is particularly noteworthy when taken in context with the reported hydrocortisone-dependent stimulation of cells entering DNA synthesis [2]. One implication of these data is that a transcription-related. hormone-dependent commitment to cell division occurs: (n) during GO of arrested cell populations: (0) during the proposed A state of cells in Gl [ 19. 201: and/or (c) during G2 of the cell cycle. Although we cannot exclude possible hydrocortisone-dependent alterations in G?, previously reported results from our laboratory indicate that hydrocortisone modulates ceI1 functions during Gl of exponentially growing cells [IS] and during the first 3-4 h after FMtHC stimulation of confluent cells [21]. Based on this information, we could speculate that hydrocortisone-dependent alterations in transcriptional activity are prerequisite to increasing the percentage of cells undergoing DNA synthesis and cell division. Alternatively, the increase in transcriptional activity could be secondary to some as yet undefined action of hydrocortisone and may only reflect an increased cell population preparing for DNA synthesis and cell division. Insight as to which of these possibilities occurs after hydrocortisone addition to WI38 cells may be obtained by comparing the kinetics of the hormone-dependent increase in transcriptional activity. the RNA products of control and hydrocortisonetreated cells and the functional capacit.ies of chromatin from control and hydrocortisonetreated cells. In regard to the kinetics of hydrocortisone-induced transcriptional alterations, there was a definite lag between hydrocortisone addition and the increase in

transcriptional activity. Superficia!!y. This suggests that hormone-dependent transcriptional alterations are secondary to the primary action of hydrocortisone and that the changes in transcriptional activity only reflect an increased number of cel!s preparing for DNA synthesis. However, it is important to mention that the delay in hydrocortisone-dependent change in transcriptional activity might be the result of our inability to detect early hormonedependent alterations in- transcriptional activity. For example, only I8-20% of the population of exponentially growing cells are in 61 phase of the cell cycle. Also, although the majority of cells in arrested confluent cultures are in GO-G 1) many of these cells may initially be unable to respond to FM+HC additions due to their location in GO-G 1. Very little information is available on the types of RNA synthesized after hydrocortisone addition. Macieira-Coelho & Loria [21] have reported the hydrocortisonedependent stimulation of [3H]uridine into ribosomes. which. presumably, indicates increased hydrocortisone-dependent ribosomal RNA synthesis. Whether these results reflect the hormone-dependent transcriptional product or are the consequence of enhanced growth of the cultures is Xclear. Alterations in chromatin functional capacity have been reported after stimulation of non-dividing WI38 cells [23]. An increased number of RNA polymerase binding sites to DNA and concurrent stimulation of transcriptional activity after addition of fresh medium to confluent, non-dividing WI38 celis has been demonstrated by Hi!! & Baserga [23]. It is possible that the increased transcriptional activity of FMtHCstimulated cultures of WI38 cells reflects an additional increase in the number of RNA

184

Ryan atld Cristofalo

polymerase-binding sites on the DNA and thus increased RNA synthesis. Rather than increasing the availability of the DNA template, hydrocortisone may stimulate transcriptional activity by increasing the catalytic activity of the nuclear RNA polymerase, by promoting additional enzyme synthesis, or by activating existing molecules. Hormonal stimulation of RNA polymerase activity has been reported in several eukaryotic tissues [24-261. For example, hydrocortisone-induced allosteric changes in RNA polymerase [27] and cortisonedependent increases in RNA polymerase I [28, 291 have been shown to result in increased ribosomal RNA synthesis in rat liver nuclei. However, our experiments showing differences in transcriptional activity using E. cofi RNA polymerase would seem to rule out possible hormonedependent endogenous RNA polymerase activation as the basis for increased transcriptional activity. In conclusion, it appears that hydrocortisone acts to modify the transcriptional activity of WI38 cells. However, whether this interaction is the primary cause in stimulating an increased percentage of cells into division, and thereby slowing down the rate of cellular senescence, or is secondary to some other action of the hormone, is unclear at present. Supported, in part, by USPHS research grants AG00034 from The National Institute of Aging and by HL-17224 from The National Heart. Lung and Blood Institute.

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Exp Cd/ Res 122 (1979)

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