©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Requirement for the Expression of Poly(ADP-ribose) Polymerase during the Early Stages of Differentiation of 3T3-L1 Preadipocytes, as Studied by Antisense RNA Induction (*)

(Received for publication, September 6, 1994; and in revised form, October 26, 1994)

Mark E. Smulson (§) Veronica H. Kang (¶) James M. Ntambi (**) Dean S. Rosenthal Ruchuang Ding Cynthia M. G. Simbulan

From the Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, D.C. 20007

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Poly(ADP-ribose) polymerase (PADPRP) is biologically significant in the rejoining of DNA strand breaks. Post confluent cultures of 3T3-L1 preadipocytes showed marked increases in PADPRP protein and activity when the cells were induced to differentiate into adipocytes. When this increase in PADPRP expression was prevented in stably transfected 3T3-L1 cells by induction of PADPRP antisense RNA synthesis, the cells did not differentiate nor undergo the two or three rounds of DNA replication that are required for initiation of the differentiation process. 3T3-Ll cells expressing PADPRP antisense RNA under differentiation conditions were easily detached from plates and in some cases eventually died. When newly expressed PADPRP protein and DNA synthesis was assessed in cells at zero time or at 24 h after induction of differentiation by incorporation of bromodeoxyuridine or [^3H]thymidine into DNA, significant incorporation was shown to occur in control cells after 24 h, but not in antisense cells. Furthermore, during the first 24 h, the co-immunoprecipitation of PADPRP and DNA polymerase alpha was observed in control cells, whereas no such complex formation was noted in the induced antisense cells, nor in uninduced control cells.


INTRODUCTION

Poly(ADP-ribose) polymerase (PADPRP), (^1)a chromatinassociated enzyme, has an absolute requirement for DNA for activity and is proportionally activated by the presence of DNA strand breaks (Smulson et al., 1977; Juarez-Salinas et al., 1979; Benjamin and Gill, 1980; Berger et al., 1980). With nuclear NAD as a substrate, PADPRP catalyzes the poly(ADP-ribosyl)ation of a subclass of nuclear proteins, including histones, high mobility group chromosomal proteins, topoisomerases (Kasid et al., 1989), protein ICP4 of herpesvirus (Blaho et al., 1992), and simian virus 40 (SV40) large T antigen (Baksi et al., 1987). PADPRP and the nuclear protein modification it catalyzes are thought to participate, together with other enzymes, in DNA repair and replication as well as in other cellular processes in which cleavage and rejoining of DNA segments may be required. These processes include cellular differentiation and transformation, sister chromatid exchange, and gene rearrangements and transpositions (Farzaneh et al., 1982; Ferro and Olivera, 1984; Ueda and Hayaishi, 1985). Support for a role of poly(ADP-ribosyl)ation in the differentiation process has been provided by a number of studies, some of which examined the effects of chemical inhibitors of PADPRP. Accordingly, DNA strand breakage, which activates PADPRP as well as the enzyme NADase, has been implicated in the terminal differentiation process of HL-60 cells (Farzaneh et al., 1987). In this regard, in HL-60 cells induced to differentiate with retinoic acid, the concentration of PADPRP mRNA increased from an initially low value and remained elevated for 12-24 h, after which the concentration declined steadily (Bhatia et al., 1990b). A similar pattern was observed during dimethyl sulfoxide-induced differentiation of HL-60 cells, with the exception that the PADPRP mRNA concentration increased later in the differentiation process (Bhatia et al., 1990b). Suzuki et al.(1989) observed a reduction in PADPRP mRNA concentration after exposure of HL-60 cells to retinoic acid for 3 days, after which time 45% of the cells showed the differentiated granulocytic phenotype. Earlier studies suggested that an increase in PADPRP activity precedes the differentiation process (Caplan and Rosenberg, 1975). In contrast, decreased PADPRP activity has been correlated with the appearance of specific gene products (Rastle and Swetly, 1978).

As mentioned above, past approaches to determining the role of PADPRP in the differentiation process depended heavily on the use of chemical inhibitors (for example, nicotinamide, thymidine, benzamide, and coumadin) (Yamagoe et al., 1991) of poly(ADP-ribose) synthesis. However, the use of these inhibitors has limitations because of their potential effects on other biological processes (Milam and Cleaver, 1984). To alleviate these problems, we have previously explored the feasibility of establishing stably transfected mammalian cells that express PADPRP antisense RNA (Ding et al., 1992). Despite the fact that the half-life of PADPRP in cells is relatively long (48-72 h), the induction of PADPRP antisense RNA in HeLa cells decreases both PADPRP activity and protein in the nucleus (Ding et al., 1992). Additionally, chromatin of PADPRP-depleted cells has an altered structure, as assayed by deoxyribonuclease I susceptibility. Interestingly, given the proposed role for PADPRP in the repair of DNA strand breaks, cells depleted of PADPRP cannot initiate DNA rejoining after strand breaks, which, as mentioned above, is a process thought to have an important role in differentiation (Farzaneh et al., 1982).

The 3T3-L1 preadipocyte cell line represents a useful tool for studying mechanisms of cellular differentiation (Mackall et al., 1976; Reed and Lane, 1980; Pekala et al., 1981). When appropriately induced by a defined hormonal treatment (insulin, dexamethasone, and methylisobutylxanthine), 3T3-L1 preadipocytes differentiate in culture into cells that possess morphological and biochemical characteristics of adipocytes (Pekala et al., 1981). A marked increase in the concentrations of lipogenic and lipolytic enzymes as well as of other adipocyte-specific proteins accompanies acquisition of the adipocyte phenotype (Ntambi et al., 1988). Depending upon the assay used, both decreases as well as increases in PADPRP activity have been reported as one of the earliest events during differentiation in these cells (Pekala and Moss, 1983; Janssen and Hilz, 1989). Accordingly, with the antisense expression conditions for HeLa cells as a guide, we have analyzed how altering the concentration of PADPRP, by antisense RNA expression, may affect the onset and progression of cellular morphological changes in 3T3-L1 preadipocytes during differentiation. Additionally, a putative role for PADPRP in apoptosis in this system is discussed.


MATERIALS AND METHODS

Vector and Probes

A 1.1-kb XhoI fragment of murine PADPRP cDNA, including 150 base pairs of 5`-untranslated sequence and the region encoding the DNA-binding domain and the amino-terminal portion of the automodification domain of PADPRP, was subcloned in the antisense orientation into the expression vector pMAM-neo (Clontech) under control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). The antisense orientation of the insert with respect to the promoter was confirmed by restriction enzyme mapping of the plasmid, which was designated pMAM-As, with BssHII, StuI, and BssHII together with SacI (see Fig. 1).


Figure 1: Structure and restriction sites of the pMAM-As plasmids containing murine PADPRP cDNA in antisense orientation downstream of the MMTV LTR. The pMAM-As expression vector contained the glucocorticoid-inducible MMTV promoter ligated to a 1.1-kb murine PADPRP 5` cDNA fragment, in reverse orientation, that encoded the DNA-binding domain and a portion of the automodification domain of the enzyme. A transcription start site within the MMTV LTR was located 260 base pairs upstream of the cloning site, and the SV40 early splicing and polyadenylation regions were located 1.0 kb downstream of the PADPRP sequence. The expected transcript size was 2.4 kb. The entire expression plasmid comprised 9.4 kb.



Southern hybridization of genomic DNA and Northern analysis of PADPRP mRNA were performed with P-labeled murine PADPRP cDNA that had been prepared by random primer extension (>10^8 cpm/µg of DNA). Antisense PADPRP RNA was detected by Northern analysis with a single-stranded RNA probe that was generated from the plasmid, pGEM-3Z (Promega). The plasmid was manipulated to contain the 1.1-kb EcoRI 5` fragment of murine PADPRP cDNA downstream of T7 promoter. The plasmid was linearized, and a P-labeled RNA probe complementary to PADPRP antisense RNA was synthesized with T7 RNA polymerase.

Cell Culture and Transfection

3T3-L1 preadipocytes (Green and Kehinde, 1974, 1975) were grown in Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 mg/ml streptomycin) (all from Life Technologies, Inc.). For transfection, 5 times 10^5 cells in 100-mm dishes were incubated overnight with 20 µg of calcium phosphate-precipitated pMAM-As DNA (Stratagene). Transfectants were selected after 48 h by culturing in medium containing G418 (0.4 mg/ml) (Life Technologies, Inc.) until colonies appeared. Fifty clones were selected and grown under continued G418 selection for further study.

DNA and RNA Analysis

Genomic DNA from 5 times 10^7 to 6 times 10^7 cells of the selected clones was extracted as described earlier (Ding et al., 1992). Twenty micrograms of DNA were digested for Southern analysis with BamHI and SacI. As expected, DNA fragments of 1.0 kb (corresponding to the MMTV LTR and murine PADPRP cDNA immediately downstream of the promoter) and 1.5 kb (corresponding to the 3` of the cDNA and a portion of the vector portion) hybridized to the mouse cDNA probe. Digestion with XhoI generated a hybridizing DNA fragment of 1.1 kb, corresponding to the entire cDNA insert.

Total RNA from 1 times 10^8 cells from each antisense cell line was isolated by guanidinium-phenol extraction (Chomczynski and Sacchi, 1987) and was analyzed by Northern analysis with the P-labeled RNA probes complementary to PADPRP antisense RNA or with P-labeled murine PADPRP cDNA.

Immunoblot Analysis

SDS-polyacrylamide gel electrophoresis and transfer of separated proteins to nitrocellulose were performed by standard procedures. Protein immobilized on nitrocellulose was stained with 0.1% Ponceau S to confirm equal transfer. Polyclonal rabbit antibodies to porcine thymus PADPRP, which have been shown to bind to murine PADPRP (Ludwig et al., 1988), were provided by Dr. Helmuth Hilz (Hamburg University). The immune complexes formed by these antibodies and PADPRP were detected with alkaline phosphataseconjugated antibodies to rabbit immunoglobulin G.

Growth Curve

Cells (5 times 10^5) were seeded on 100-mm dishes and, 1 day later, incubated in the presence or absence of dexamethasone (1 µM). Samples were collected at various times, and the cell number and viability determined by cell counting with a Coulter counter and with a hemocytometer in the presence of trypan blue.

Induction of Antisense RNA Synthesis

Culture dishes (75 cm^2) were inoculated with 1 times 10^5 cells. The following day, cells were incubated in DMEM supplemented with 10% FBS and dexamethasone (1 µM). Cells were collected for analysis at various times.

Induction of Differentiation of 3T3-L1 Preadipocytes

Culture dishes (75 cm^2) were inoculated with 1 times 10^5 control 3T3-L1 cells or stably transfected 3T3-L1 cells, and the cells were grown to confluence in DMEM with 10% FBS. After reaching confluency, the cells were maintained for an additional 2 days. To initiate differentiation, we supplemented the DMEM with methylisobutylxanthine (0.5 mM), dexamethasone (1 µM), insulin (1.7 µM), and 10% FBS. After 48 h, the medium was replaced with DMEM supplemented with insulin (1.7 µM) and 10% FBS. After a further 48 h, the medium was replaced with DMEM supplemented with only 10% FBS.

Oil Red O Staining for Triglyceride Droplets

Cells were washed twice with phosphate-buffered saline (PBS) and then fixed with 3.7% formaldehyde in PBS for 10 min. The cells were washed twice with deionized water, incubated with Oil Red O dye (0.3%) for 1 h at room temperature, rinsed with water, and then observed by phase-contrast light microscopy.

PADPRP Activity Assay

Cells (10^6) were harvested by scraping, washed three times with ice-cold PBS, and sonicated three times with 10-s bursts in order to break cells and introduce an excess of DNA strand breaks (required for PADPRP activity). The initial velocity of [P]NAD incorporation into acid-insoluble acceptors was measured at 25 °C for 1 min, according to the method described by (Cherney et al., 1985).

Immunocytochemical Methods

Cells at zero time or 24 h after induction of differentiation were pulsed with 10 µM bromodeoxyuridine (BrdU) for 1 h, washed twice with ice-cold PBS, fixed with cold 95% ethanol for 30 min at room temperature, and subsequently denatured with 0.07 M NaOH in 70% ethanol for 2 min. After washing twice with PBS, the cells were incubated for 4 h in a humid chamber with both mouse monoclonal anti-BrdU (Becton Dickinson) and anti-PADPRP, diluted 1:10 and 1:1000, respectively, with PBS containing 12% bovine serum albumin, followed by a final incubation in the dark with a mixture of rhodamine-labeled anti-mouse (Tago, 1:10) and goat fluorescein-labeled anti-rabbit (1:40). Between antibody incubations, the cells were washed twice with PBS and once with water. The plates were then cut and mounted with Vectashield, and observed under a Zeiss immunofluorescence microscope.

DNA Replication Assay by [^3H]Thymidine Incorporation

Cells at indicated time intervals after induction of differentiation were harvested by scraping, aliquoted into cell wells (10^6 cells in 2 ml of medium/well), allowed to stabilize in a 37 °C CO(2) incubator for 1 h, and then pulsed for 15 min with [^3H]thymidine (TdR, 40 Ci/mmol, 0.2 µCi/ml). The cells were then collected by centrifugation, washed extensively and resuspended in PBS, and lysed with 0.1% SDS and 1 mM EDTA. Measurement of acid-insoluble radioactivity was performed by trichloroacetic acid precipitation on GF/C filters (Whatman), followed by washes with 20% trichloroacetic acid, 70% ethanol, and 95% ethanol and liquid scintillation counting of the filters.

Immunoprecipitation and Immunoblotting

Immunoprecipitation was performed with slight modifications as described previously for these two enzymes (Simbulan, et al., 1993). Cells were washed twice with ice-cold PBS and lysed with 1 ml of EBC buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40, and 0.1 trypsin inhibitory unit of aprotinin) for 20 min on ice. The lysates were then clarified by centrifugation, aliquoted to 200 µl/sample (50 µg protein), and pre-cleared with 10 µl/sample of protein A-Sepharose overnight at 4 °C. After centrifugation, the supernatants were rocked for 1 h with 0.5 ml NET-N buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40) containing a mouse monoclonal anti-DNA polymerase alpha antibody (ATCC cell line SJK-132-20, 10 µl of ascitis/sample), and rocked for another 20 min with 20 µl of a 1:1 mixture of protein A-Sepharose, freshly washed and suspended in Tris-buffered saline with 10% bovine serum albumin. The beads were washed five times with 1 ml of NET-N buffer, and the immunoprecipitates on the beads were subsequently separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. After staining with Ponceau S stain to confirm transfer, the blots were blocked, incubated with polyclonal rabbit anti-PADPRP (1:1000), probed with peroxidase-labeled anti-rabbit IgG, and detected by electrochemiluminescence.


RESULTS

PADPRP Antisense Vector and Cell Transfection

With a human PADPRP cDNA probe, murine genomic and cDNA partial clones encoding PADPRP were isolated previously (Huppi et al., 1989). A 1.1-kb fragment of murine cDNA, encoding the DNA-binding domain and the amino-terminal portion of the automodification region of PADPRP, was subcloned in the antisense orientation into the expression vector pMAM-neo under control of the MMTV promoter (Fig. 1). The antisense orientation of the PADPRP cDNA in the resulting plasmid, pMAM-As, was verified by restriction enzyme mapping. The pMAM-As construct was stably transfected into 3T3-L1 preadipocytes by calcium phosphate precipitation. After selection in G418 for 4 weeks, 50 resistant colonies were isolated and screened for decreased PADPRP activity after incubation with dexamethasone, as described previously for HeLa cells (Ding et al., 1992). DNA was prepared from seven of the cell lines and analyzed for integration of the antisense PADPRP sequence. The 1.1-kb antisense cDNA fragment was detected after digestion of the DNA with XhoI in all seven cell lines (not shown). The hybridization intensity corresponding to the 1.1-kb fragment was significantly greater than that observed for the endogenous murine PADPRP gene, suggesting that pMAM-As was integrated in high copy numbers.

Expression of PADPRP Antisense RNA in 3T3-L1 Cells and Effects on Endogenous Transcripts

To characterize and quantify the kinetics of antisense PADPRP RNA expression, we performed Northern analysis with control 3T3-L1 cells and two transfected 3T3-L1 cell lines (As 5 and As 6) after incubation with dexamethasone for various times (Fig. 2). Blots were probed with a riboprobe that hybridizes specifically to murine PADPRP antisense RNA (Fig. 2B). An antisense PADPRP RNA of 2.4-kb was detected in clones As 5 and As 6 within 5 h after induction by dexamethasone; thereafter, the amount of the antisense RNA decreased, but was still detectable after 72 h (Fig. 2A). The antisense RNA included a portion of the MMTV promoter region upstream of the PADPRP cDNA insert, as well as SV40 sequences downstream of the PADPRP cDNA (Fig. 1). Antisense RNA was not detected in the As 5 and As 6 cell lines in the absence of dexamethasone (Fig. 2A) indicating that the MMTV promoter was under tight control in these cells. In addition, expression of a hybridizing antisense transcript was not observed in control cells in the absence or presence of dexamethasone (Fig. 2A). The decrease in the amount of PADPRP antisense RNA in cell lines As 5 and As 6 between 5 and 72 h after dexamethasone treatment is consistent with several observations showing that antisense RNA is unstable and that hybrid sense-antisense duplex RNAs have short half-lives (Izant, 1984).


Figure 2: Dexamethasone-induced synthesis of PADPRP antisense RNA in 3T3-L1 cells stably transfected with plasmid pMAM-As. A, control (nontransfected) 3T3-L1 cells and 3T3-L1 cells stably transfected with pMAM-As (lines As 5 and As 6) were incubated with dexamethasone (1 µM) for the indicated time periods. Total RNA was isolated, and 10 µg were subjected to Northern analysis as described under ``Materials and Methods.'' B, Northern blots were hybridized with a P-labeled single-stranded RNA probe that was synthesized from a pGEM-3Z vector containing the 1.1-kb mouse PADPRP cDNA fragment used to construct pMAM-As. The riboprobe was synthesized in the sense orientation with T7 RNA polymerase.



The influence of dexamethasone-induced PADPRP antisense RNA on the concentration of endogenous PADPRP mRNA in cell lines As 5 and As 6 was determined by Northern analysis with a murine PADPRP cDNA probe (data not shown). In control 3T3-L1 cells, treatment with dexamethasone for up to 72 h had no effect on the steady-state concentration of PADPRP mRNA. In contrast, treatment of As 5 and As 6 with dexamethasone at various time points reduced the concentration of PADPRP mRNA by 80-95%.

Effect of Induction of PADPRP Antisense RNA on 3T3-L1 PADPRP Concentration

Total cellular protein was extracted from antisense cell lines As 5 and As 6 as well as from control cells that had been incubated with dexamethasone for various periods of time. Proteins were analyzed by immunoblotting with polyclonal antibodies to porcine PADPRP (Fig. 3); these antibodies were previously shown to react with a 113-kDa protein, corresponding to full-length PADPRP, as well as with 89- and 90-kDa proteins, which correspond to truncated PADPRP molecules observed in murine cell extracts, not present in other species examined (Ludwig et al., 1988). Because of the additional PADPRP-derived degradation products, some variation in the number of immunostained bands were occasionally encountered in contrast to earlier studies with human cells (Ding et al., 1992). A Ponceau stain of the immunoblot indicated that equivalent amounts of protein were loaded onto the gel in each lane and that there was no overall cellular protein degradation during PADPRP antisense RNA induction (data not shown).


Figure 3: Effect of PADPRP antisense RNA synthesis on cellular PADPRP concentration. Control (nontransfected) 3T3-L1 cells (A) and As 5 and As 6 cells (B) were incubated with dexamethasone (1 µM). At the indicated times, cells were washed with PBS, and their protein concentration was determined. Equal amounts of total cellular protein (50 µg) were subjected to electrophoresis on an SDS-polyacrylamide gel, and the separated proteins were then transferred to a nitrocellulose filter. The filter was incubated with rabbit antiserum to porcine thymus PADPRP at a dilution of 1:2000. No Dex, cells incubated in the absence of dexamethasone. The positions of molecular mass standards (STD) are indicated.



In control cells, incubation with dexamethasone for 72 h did not affect PADPRP concentration (Fig. 3A). Furthermore, the amounts of PADPRP in uninduced As 5 and As 6 cells were approximately the same as that in control cells. In contrast, the concentration of PADPRP was markedly reduced in As 5 and As 6 cells after induction of PADPRP antisense RNA synthesis. The PADPRP concentration in these cells decreased by 40, 60, and 80% after 48, 72, and 96 h of exposure to dexamethasone, respectively (Fig. 3B). The 113-kDa full-length PADPRP protein and the truncated 89-kDa protein decreased proportionally with induction of antisense RNA synthesis. To verify that the decrease in PADPRP concentration was not attributable to decreased growth rate, we showed that the amounts of PADPRP in cells incubated for 0 and 72 (As 6) or 96 (As 5) h in the absence of dexamethasone were virtually identical (Fig. 3B).

Effect of Antisense RNA Synthesis on PADPRP Activity

PADPRP activity was measured directly in sonicated extracts of control and antisense cells at various times after induction by dexamethasone. The sonication procedure had previously been established to maximize the number of endogenous DNA strand breaks, which are required for PADPRP activity. In the PADPRP assay, activity is proportional to the number of PADPRP molecules because automodified PADPRP is the major product of the reaction (Cherney et al., 1985). PADPRP activity decreased by 50-60% in As 5, As 6, and As 7 cells after 48 h of dexamethasone treatment (Table 1). A maximal decrease in activity of 75% was observed after 72 h of exposure to dexamethasone. The extent of inhibition of PADPRP activity was essentially consistent with both the corresponding decreases in the concentrations of PADPRP protein (Fig. 3) and PADPRP mRNA (data not shown).



Effect of Differentiation of 3T3-L1 Preadipocytes on the Concentration of PADPRP

Several studies have shown that PADPRP activity changes during the differentiation of 3T3-L1 preadipocytes into adipocyte-like cells. However, the observed patterns of changes have differed, depending on the nature of the polymerase assay used (Pekala et al., 1981; Lewis et al., 1982; Pekala and Moss, 1983; Janssen and Hilz, 1989). Because of these differences, we have reexamined the effect of 3T3-L1 cell differentiation on PADPRP by both activity measurement as well as immunoblot analysis. Control 3T3-L1 cells were allowed to grow for 2 days after reaching confluency, and differentiation was then initiated by exposure to insulin, dexamethasone, and methylisobutylxanthine. Cells were collected daily and examined for both morphological and biochemical characteristics of adipocytes. The cells progressed through two additional rounds of cell division, which is a prerequisite for differentiation (Villarreal, 1991). After 2 days, differentiating cells adopted an overall spherical morphology and after 3 days synthesized triglyceride droplets (see below). A transient 2-fold increase in PADPRP activity was apparent during the first 2 days of differentiation (Fig. 4).


Figure 4: PADPRP activity and concentration in control 3T3-L1 preadipocytes during differentiation. Two days after achieving confluency, 3T3-L1 cells were induced to differentiate with dexamethasone, methylisobutylxanthine, and insulin as described under ``Materials and Methods.'' At the indicated times, cells were assayed for PADPRP activity by the sonication method, as described under ``Materials and Methods,'' with 10-20 µg of protein for each determination. Values are the means of duplicate determinations. Inset, immunoblot analysis of PADPRP concentration. Equal amounts (50 µg) of total cellular protein were subjected to immunoblot analysis as described in Fig. 3. The arrow indicates the 113-kDa band corresponding to the full-length murine PADPRP.



The catalytic activity of PADPRP is influenced by the accessibility of acceptor molecules, and is also linearly dependent on the number of DNA strand breaks and effected by several other properties of chromatin (Butt et al., 1978, 1979; Benjamin and Gill, 1980; Berger et al., 1987; Jacobson and Jacobson, 1989). Because of these potential complications, we also examined the effect of differentiation on PADPRP by immunoblot analysis, which had not previously been examined. A marked increase in immunoreactive PADPRP was observed during the first 2 days of differentiation (Fig. 4, inset). Variability in the number of immunoreactive bands observed may be due to the fact that PADPRP has been reported to degrade in cells into peptides of discrete sizes (Hotlund et al., 1983; Surowy and Berger, 1985), which are also detectable with the porcine anti-PADPRP antibody. PADPRP activity decreased to below control values by 4 days, as the cells terminally differentiated into adipocytes. At the fully differentiated state (day 8), immunoreactive PADPRP was virtually undetectable. These results are consistent with previous studies showing low PADPRP activity in terminally differentiated cells (Cherney et al., 1985).

Effect of Reduced PADPRP Activity on Preadipocyte Growth, Morphology, and Differentiation

To investigate whether decreased PADPRP activity as a result of PADPRP antisense RNA induction would trigger 3T3-L1 differentiation, we exposed control cells and antisense cell lines As 5 and As 6, 2 days after achieving confluency, to dexamethasone. Dexamethasone had no effect on control 3T3-L1 cell morphology and the cells remained quiescent. Furthermore, dexamethasone treatment alone did not induce As 5 and As 6 cells to differentiate. However, in the presence of dexamethasone, As 5 and As 6 cells appeared spindle-shaped, and the boundaries between cells were clearly discernible; in contrast, the membrane processes of control cells, as well as of As 5 and As 6 cells in the absence of dexamethasone, overlapped, and the boundaries between cells were not visible (data not shown).

The effect of PADPRP antisense RNA was subsequently examined during differentiation (Fig. 5). The marked increases in PADPRP activity and protein observed during the first 2 days of differentiation of control 3T3-L1 cells (Fig. 4) were not apparent in antisense cells; PADPRP activity and protein remained constant (Fig. 5). A slightly higher immunological reaction of the truncated PADPRP was noted at day 2, although this was not associated with increased PADPRP at this time point. Furthermore, in contrast to control 3T3-L1 cells (both nontransfected and transfected with pMAM-neo only), As 5 and As 6 cells did not accumulate cytoplasmic triglyceride as visualized by phase-contrast microscopy and oil red O staining (Fig. 6). In both control cell lines, triglycerides were observed from day 3 of differentiation and were visible in almost 90% of the cells by day 7. As 5 and As 6 cells also did not exhibit any of the morphological changes observed for control cells after induction of differentiation.


Figure 5: PADPRP activity and concentration in 3T3-L1 As 5 cells after incubation with inducers of differentiation. Two days after achieving confluency, 3T3-L1 cells were exposed to inducers of differentiation. At the indicated times, cells were assayed for PADPRP activity by the sonication method. Values are the means of duplicate determinations. Inset, equal amounts (20 µg) of total cellular protein were subjected to immunoblot analysis of PADPRP as described in Fig. 4. The arrow indicates the 113-kDa band corresponding to the full-length murine PADPRP.




Figure 6: Cytoplasmic triglyceride accumulation in control and pMAM-As-transfected 3T3-L1 cells. Seven days after exposure to inducers of differentiation, cells were stained with Oil Red O dye and examined by phase-contrast microscopy. A control (nontransfected) 3T3-L1 cells. B, mock-transfected 3T3-L1 cells (cells with transfected with pMAM-neo vector devoid of PADPRP cDNA. C, As 5 cells; D, As 6 cells.



These antisense cells did not undergo the normal two or three rounds of cell division during the initial 2 days after exposure to inducers of differentiation (Fig. 7). Furthermore, the cells became easily detached from plates after 5 days of exposure to differentiation inducers and eventually died, as revealed by their failure to exclude trypan blue. In some instances, the antisense cells progressed through one round of cell division (Fig. 8, inset); however, this process resulted in even more extensive cytotoxicity. These results are consistent with a potential role of PADPRP in apoptosis (Nosseri et al., 1994; Kaufmann et al., 1994) and antisense 3T3-L1 cells might prove useful to further study this important biological process. Although the role of PADPRP in cells that undergo apoptosis is still unclear, it has been observed that inhibitors of PADPRP may either enhance or reduce the extent of apoptosis (Rice et al., 1992, Nosseri et al., 1994). Confluent antisense cells, treated with dexamethasone alone, did not undergo cell death, even after 2 weeks of exposure to this agent. Thus, the cytotoxicity observed in the antisense cell lines, exposed to differentiation inducers, was probably not attributable to antisense expression per se. The expression of several other antisense transcripts in 3T3-L1 cells, such as those to actin or ferritin heavy chain, has been shown not to affect differentiation or to have adverse effects on growth (Wenz et al., 1992).


Figure 7: Proliferation of control and pMAM-As-transfected 3T3-L1 cells after incubation with inducers of differentiation. Two days after achieving confluency, control (nontransfected) 3T3-L1 cells and antisense cells were incubated with inducers of differentiation as described under ``Materials and Methods.'' Cells were collected at the indicated times and counted with a hemocytometer and trypan blue dye and with a Coulter counter.




Figure 8: Time course of DNA replication in 3T3-L1 control and antisense cells during differentiation, as measured by the rate of [^3H]-TdR incorporation into DNA. Post-confluent control and antisense cells were induced to differentiate, harvested at indicated time intervals, pulse labeled for 15 min with [^3H]TdR (0.2 µCi/ml), and the acid insoluble radioactivity was then measured by TCA precipitation and liquid scintillation counting as described under ``Materials and Methods.''



DNA Synthesis During the Initial Stages of Differentiation

Based upon the results on cell proliferation shown in Fig. 7, the rate of DNA replication during the early stages of differentiation was measured in samples pulse labeled for 15 min with [^3H]TdR (Fig. 8). In control cells, DNA synthesis is initiated after approximately 12 h after induction of differentiation, peaked at 24 h, and declined to 30% or less by 30 h. In contrast, negligible [^3H]TdR incorporation was noted in antisense cells during the same time frame.

Nuclear Co-localization of Induced PADPRP and DNA Synthesis by Immunocytochemical Staining

To further examine the expression of PADPRP and the onset of DNA replication (Fig. 8), we compared the cellular localization of PADPRP and incorporation of BrdU into newly replicated DNA after 1 day of differentiation by immunocytochemical staining (Fig. 9). In control cells, prior to differentiation (zero time), there was only limited incorporation of both BrdU and PADPRP protein expression observed (Fig. 9A); however, after 24 h of induction of differentiation, there was a significant incorporation of BrdU in the nuclei of control cells, consistent with the cell division data presented in Fig. 7and the [^3H]TdR incorporation data (Fig. 8). Concomitantly, there was a marked expression of PADPRP protein localized to the nuclei of control cells 24 h after induction of differentiation, also consistent with Western data presented in Fig. 4.


Figure 9: Determination of BrdU incorporation and expression of PADPRP during the first 24 h of differentiation in intact cells by immunocytochemical methods. Two days after achieving confluency, control (non-transfected) 3T3-L1 cells (A) and antisense cells (B) were incubated with inducers of differentiation for 24 h. Selected nuclei from A were enlarged (C). Cells were collected at zero time (prior to adding differentiation inducers) and 24 h after induction of differentiation as described under ``Materials and Methods.'' For BrdU incorporation, the cells were incubated with 10 µM BrdU for 1 h and immunodetected by a mouse monoclonal anti-BrdU, followed by rhodamine-labeled anti-mouse IgG. To detect PADPRP immunologically, fixed cells were incubated with rabbit anti-PADPRP, followed by fluoresceinlabeled anti-rabbit IgG, as described under ``Materials and Methods.''



Selected nuclei from Fig. 9A were enlarged (Fig. 9C). Both BrdU and PADPRP appear to be concentrated in distinct intranuclear granular foci, although it is not clear if they represent similar substructures of the nuclei. As evident in Fig. 9B, there was negligible BrdU and PADPRP immunocytochemical staining in nuclei of antisense 3T3-L1 cells after 24 h of induction of differentiation.

Association of DNA Polymerase alpha with PADPRP During Differentiation

To initiate studies on PADPRP's role(s) in biological events occurring during the early stages of differentiation, we studied the interaction of PADPRP with DNA polymerase alpha (DNA pol alpha). This approach was suggested by earlier data showing that purified PADPRP binds to and stimulates the activity of immunoaffinity-purified calf thymus or human DNA pol alpha by about 10-60-fold in a dose-dependent manner (Simbulan et al., 1993). The effect is quite specific since the presence of PADPRP has no such effect on the in vitro activities of DNA polymerases beta, , , and , as well as DNA primase. The stimulation of DNA pol alpha activity appears to be due to direct PADPRP binding, since a complex of PADPRP and DNA pol alpha co-immunoprecipitates with monoclonal DNA pol alpha antibody (Simbulan et al., 1993).

In the experiment in Fig. 10, both control and antisense cells were examined for interaction of PADPRP and DNA pol alpha at zero time and 24 h after induction of differentiation. Crude extracts derived from these cells were immunoprecipitated with a monoclonal antibody to DNA pol alpha. The immunocomplex was then isolated by addition of protein A-Sepharose beads and separated by SDS-gel electrophoresis, followed by Western blotting using antibody to PADPRP.


Figure 10: PADPRP and DNA polymerase alpha associate during early stages of differentiation, as determined by co-immunoprecipitation. Both control and antisense cells were treated essentially as described in the legend to Fig. 9. At zero time (prior to differentiation) and 24 h after induction of differentiation, cells were collected, washed, lysed, and aliquots of the cell extracts (50 µg of protein) were immunoprecipitated with a monoclonal anti-DNA pol alpha antibody, as described under ``Materials and Methods.'' The immunocomplex was then separated by SDS-gel electrophoresis, transferred to nitrocellulose by Western transfer, probed with a rabbit anti-PADPRP antibody, and detected by electrochemiluminescence. Lanes 1, 3, 4, 7, and 8 are samples from control cells, at zero time (lane 1), or 24 h after induction of differentiation (lanes 3, 4, 7, and 8); lanes 3 and 4 are duplicate determinations. In lane 7, control cell extracts were incubated with only protein A-Sepharose, prior to electrophoresis, and, in lane 8, preimmune serum was substituted for the anti-DNA pol alpha antibody. Lanes 1, 5, and 6 represent samples from antisense cells, either at zero time (lane 1) or at 24-h differentiation (lanes 5 and 6); lanes 5 and 6 are duplicate determinations.



With control cells at zero time (Fig. 10, lane 1), PADPRP was not observed to co-immunoprecipite with DNA pol alpha, consistent with the low levels of PADPRP in cells prior to differentiation (Fig. 4). However, after 24 h of induction of differentiation (lanes 3 and 4) binding of DNA pol alpha and PADPRP was observed, as shown by the immunostained band for murine PADPRP at 113 kDa. Co-immunoprecipitation of the two enzymes did not occur if only protein A-Sepharose was present (lane 7), nor if preimmune serum replaced anti-DNA pol alpha (lane 8). As anticipated, we did not observe the co-immunoprecipitation of PADPRP with DNA pol alpha in antisense cells, either at zero time or after 24 h of differentiation (lanes 2, 5, and 6). The data in Fig. 8Fig. 9Fig. 10further support our earlier results using purified enzymes (Simbulan et al., 1993) and suggest that PADPRP and DNA pol alpha may associate at some stage during the round of DNA replication required for differentiation.


DISCUSSION

Although chemical inhibitors have proven useful in investigating potential functions of PADPRP (Caplan et al., 1979; Morioka et al., 1979; Brac and Ebisuzaki, 1985) they are not without potential complications (Milam and Cleaver, 1984). Thus, we have applied a more specific approach, that of antisense RNA synthesis, to determine the effect of inhibition of poly(ADP-ribosyl)ation on the differentiation of 3T3-L1 preadipocytes. Both PADPRP protein and activity show a transient, marked increase on exposure of cells to inducers of differentiation (Fig. 5). The increase in PADPRP expression was prevented in this study by antisense RNA synthesis. Under these conditions, both cellular proliferation and differentiation did not occur ( Fig. 6and Fig. 7). The synthesis of antisense RNA corresponding to beta-actin or to ferritin heavy chain has previously been shown to have no influence on the differentiation of these cells (Wenz et al., 1992). Alternatively, Fang-Tysr and Lane(1992) have shown that antisense expression of an RNA associated with adipocyte differentiation (C/EBP) prevented expression of a number of adipocyte specific mRNAs and also prevented differentiation. The observation that the increases in both PADPRP protein and activity occur at the same time as cellular proliferation at the onset of differentiation does not directly prove a correlation between these two events. However, the increased stability of PADPRP mRNA during DNA replication (Bhatia et al., 1990a), as well as the high concentrations of poly(ADP-ribosyl)ated intermediates at the middle and end of S phase (Kidwell and Mage, 1976; Wong et al., 1983), supports the concept that the increase in PADPRP expression during the early stages of differentiation may be critical for the differentiation of preadipocytes because of a role of the enzyme in a stage in the cell cycle prior or during DNA replication.

Various models, all of which involve chromatin restructuring, have been proposed to explain the necessity for DNA replication prior to differentiation (Villarreal, 1991). Implicit in many of these models is the repositioning or alteration of nucleosomes, which may result in the activation or inhibition of specific genes and may involve various nuclear protein modifications, including poly(ADP-ribosyl)ation. In this regard, poly(ADP-ribosyl)ation may aid in either relaxation (de Murcia et al., 1986) or condensation of chromosomal proteins around the replicating regions of chromatin (Butt et al., 1980).

A role for poly(ADP-ribosyl)ation in the regulation of chromatin structure is supported by various previous observations. In HeLa cells synthesizing PADPRP antisense RNA, we observed a hypersensitivity of chromatin to deoxyribonuclease 1 (Ding et al., 1992). Chromatin regions corresponding to replicating regions of DNA shows transient hypersensitivity to nuclease digestion (Smerdon, 1989), which may be related to localized changes in nuclear organization of the 300-Å fiber of chromatin. Additional experimental systems have indicated that poly(ADP-ribosyl)ation of nucleosomal proteins may influence the structure of chromatin and thereby facilitate DNA replication and other nuclear processes (Jump et al., 1980; Althaus et al., 1985; Chatterjee et al., 1989; Jacobson and Jacobson, 1989). Recently, Mathis and Althaus(1986) showed a significant influence of poly(ADP-ribosyl)ation and chromatin structure on DNA repair: in the presence of chemical inhibitors of PADPRP, DNA adducts were inaccessible for repair and tended to accumulate in nonnucleosomal regions of chromatin.

The initial stages in the rejoining of DNA strand breaks were shown to be inhibited in HeLa cells depleted of PADPRP as a result of PADPRP antisense RNA synthesis (Ding et al., 1992). Thus, the low viability of 3T3-L1 cells induced both to differentiate and to synthesize PADPRP antisense RNA may result from a toxic accumulation of Okazaki fragments generated during the early stages of differentiation. In this regard, Lonn and Lonn(1988) showed that the accumulation of unligated Okazaki fragments is increased significantly in human cells incubated with the PADPRP inhibitor, 3-aminobenzamide.

PADPRP contains two zinc fingers, which are required for the binding of the enzyme to both single-strand and double-strand DNA breaks in a sequence-independent manner (de Murcia et al., 1983; Cherney et al., 1987). Furthermore, the catalytic activity of PADPRP is dependent on DNA strand breaks. The replication forks associated with differentiation-related DNA synthesis contain numerous free ends that could activate the poly(ADP-ribosyl)ation of nucleosomal proteins, which, in turn, might alter the structure of these regions. In this regard, Satoh and Lindahl(1992) have proposed a model, on the basis of in vitro data, in which non-poly(ADP-ribosyl)ated PADPRP binds to ends of DNA during DNA replication or repair. Subsequently, the large negative charge resulting from extensive automodification of bound PADPRP would promote removal of the enzyme from the DNA ends to allow DNA ligation. This proposed mechanism is in agreement with our previous data showing that the addition of NAD (the substrate of PADPRP) to chromatin in vitro increases the accessibility of either endogenous or exogenously added DNA polymerase to DNA primers (Roberts et al., 1974). In further support of the putative association of poly(ADP-ribosyl)ation with DNA synthesis is the observation that the replicating regions of chromatin are selectively retained relative to nonreplicating regions, when either polyoma or SV40 minichromosomes are subjected to anti-poly(ADP-ribose) antibody affinity chromatography (Baksi et al., 1987).

We recently tested the above model with bacterially expressed mutants of PADPRP with deletions in the three major functional domains of the enzyme (Smulson et al., 1994). Deletion mutants with an intact amino-terminal DNA binding domain inhibit DNA repair, whereas a mutant with a deletion in the DNA-binding domain does not inhibit the in vitro assay. However, whether the deletion is in the NAD-binding, active site domain, or the automodification domain, the inhibition of repair exerted by these mutant proteins is not alleviated by NAD.

In the current work, we have provided data showing the co-immunoprecipitation of PADPRP and DNA pol alpha in crude extracts derived from control cells during the initial stages of differentiation, but this was not observed in the antisense cells (Fig. 10). In this regard, we previously showed that PADPRP binds to purified calf thymus DNA pole alpha and stimulates its activity by about 10-60-fold in a dose-dependent manner. It was also observed that the stimulatory activity produced by this physical association of PADPRP and DNA pol alpha is lost when PADPRP is automodified. Apparently, in the presence of PADPRP, the saturation curve for the DNA template primer becomes sigmoidal; at very low concentrations of DNA, PADPRP inhibits the reaction in competition with template DNA, while at higher DNA doses the reaction is significantly stimulated by increasing the V(max) of the reaction, which suggests an allosteric effect of PADPRP on the activity of DNA pol alpha (Simbulan et al., 1993).

Reddy and Pardee(1980) provided interesting data earlier and Reddy and Fager(1993), more recently, which identify a nuclear complex of ``DNA precursor-synthesizing enzymes juxtaposed with the replication apparatus comprising DNA polymerase, other enzymes and structural proteins.'' By immunofluorescent imaging analysis, Li et al.(1993) further showed that DNA pol alpha and DNA ligase, both of which exists in a 21 S multienzyme complex for DNA synthesis, can be localized to distinct ``granular-like foci,'' whereas DNA pol beta, not associated with the complex, appears to be diffusely distributed in the nucleus. We have also previously shown that, in crude extracts of calf thymus, a portion of PADPRP exists in a 400-kDa as well as a large 700-kDa complex, containing DNA pol alpha (Simbulan et al., 1993). We anticipate that the 3T3-L1 antisense cell lines characterized in our current study may help facilitate a better understanding of the significance of the association of PADPRP and DNA pol alpha and also what other biological roles poly(ADP-ribosyl)ation may participate in during differentiation.


FOOTNOTES

*
This work was supported in part by National Cancer Institute Grant CA25344 and by funding from the United States Air Force Office of Scientific Research (AFOSR-89-0053). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Georgetown University School of Medicine, Dept. of Biochemistry and Molecular Biology, Basic Science Bldg., Rm. 351, 3900 Reservoir Rd., N.W., Washington, DC 20007. Tel.: 202-687-1718 and 1089; Fax: 202-687-7186.

Work submitted to the Department of Biochemistry and Molecular Biology in partial fulfillment of the requirements for the Ph.D. degree.

**
Supported by United States Public Health Service Grant DK-42825. Present address: University of Wisconsin, Dept. of Biochemistry, Henry Mall 420, Madison, WI 53706-1569.

(^1)
The abbreviations used are: PADPRP, poly(ADP-ribose) polymerase; MMTV, mouse mammary tumor virus; LTR, long terminal repeat; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BrdU, bromodeoxyuridine; TdR, thymidine deoxyribose; pol alpha, polymerase alpha; kb, kilobase pair(s).


ACKNOWLEDGEMENTS

We thank Jane Murphy for help with the experiments, Dr. Helmuth Hilz, University of Hamburg, for the generous gift of antibody to murine PADPRP, and Dr. Shonen Yoshida, Nagoya University, for the antibody to DNA pol alpha.


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