Prothymosin at mRNA Levels Are Invariant throughout the Cell Cycle*

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 267,No. 12. Issue of April 25, pp. 8692-8695, 1992 Printed in U.S.A.

0 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Prothymosin at mRNA Levels Are Invariant throughout the Cell Cycle* (Received for publication, November 8,1991)

Juan B. Zalvide, Esperanza CancioS, ClaraV. Alvarez, Benito J. RegueiroSp, and Fernando Dominguezn From the Departamento de Fiswlogia, Laboratorio de Neurociencias Ramon Dominguez,the $Departamento de Microbiologia and $Hospital Xeral de Galicia, Faeultad de Medicinn, Santiago de Compostela, Spain

Prothymosin a (PT-a) mRNA levels were evaluated at different stages during the cell cycle.NIH 3T3 cells were synchronized: (a)by serum deprivation, ( b ) by mitotic shake off after nocodazole arrest, and (c) by double thymidine block. Cell synchronism was estimated by flow cytometry. In cells grown inserum-free medium, PT-a mRNA levels werealmost undetectable. 14 h after serum restoration PT-a mRNA was induced as had been described by others (Eschendfeldt, W. H., and Berger, S. L. (1986) Proc. Natl. Acad. Sci. U.S. A. 83, 9403-9407). PT-a mRNA induction seems to require the synthesis of proteic factor(s) since PT-a mRNA response to serum restoration was abolished in the presence of cycloheximide. Interestingly, cycling cells that were synchronized at different stages of the cycle by means of mitotic shake off after nocodazole arrest or double thymidine block did not show variations in the levels of PT-a mRNA when progressed synchronously through the cycle. On the contrary, histone H4 mRNA was expressed only during the S phase. These data indicate that PT-a mRNA was present in roughly the same amount throughall phases of the cell cycle, arguing against the concept that PT-a is a cell cycle-regulated gene.

Prothymosin a (PT-a)’is a small highly acidic protein of unknown function, first isolated as theprecursor of thymosin a1( l ) , a peptide that was supposed to be a thymic hormone. PT-a is believed to be the endogenous peptide from which thymosin a1is formed by proteolytic modification during the tissue extraction procedure (1). Recent evidence showed that PT-a is not secreted, because it is synthesized without the formation of a larger precursor polypeptide containing the hydrophobic signal sequence (2, 3), and its mRNA is translated in free ribosomes, instead of endoplasmic reticulum-bound ribosomes, as wouldbe expected from a secreted peptide (4).Little is known about the nature of the intracellular function of PT-a but, although it has also been found in the cytoplasm (5, 6), PT-a has more consistently been found in the nucleus, leading to the assumption that it has a nuclear site of action (7-11). The finding that PT-a mRNAwas induced in serum-deprived NIH 3T3 cells when they were stimulated to proliferate (3)

* This work was supported in part by Grant XUGA84202888 from the Xunta de Galicia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. 1To whom correspondence and reprint requests should be addressed. The abbreviations used are: PT-a, prothymosin a; SDS, sodium dodecyl sulfate.

strongly suggested that PT-a expression in a cell is related to its proliferative activity. Laterreports showed that PT-a mRNA expression was correlated to theproliferative activity of T cells (12), and that thymosin a1 immunoreactivity was correlated to the proliferative state of a small intestine-derived cell line (9). Immunohistochemical studies have also shown that in various tissues from the rat, PT-a is expressed in proliferating and not in quiescent cells; this pattern of expression is common to all tissues studied so far (13-15). PT-a gene is induced by myc (16), the earliest discovered and perhaps the most prominent nuclear oncogene that has been implicated in the control of normal cell proliferation and the transformation of many cell lineages, further implying that PT-a plays a role in cellular proliferation. Direct evidence supportingthis concept was obtained when PT-a mRNA antisense oligomerswere shown to inhibit cell division in myeloma cells (17). Based on these experiments, the moment of action of PT-a during the cell cycle has been tentatively placed in Gz(17). Nevertheless, it has recently been proposed that PT-a expression is not associated to cell proliferation (18). To further clarify the putative role of PT-a in cell proliferation, we decided to study PT-a expression in proliferating and cyclingcells, since regulatory mechanisms in actively cycling cells are entirely different from those that mediate growth in quiescent cells stimulated to proliferate (19-21). MATERIALS ANDMETHODS

Cell Culture and Synchronization Procedures-Mouse NIH 3T3 cells were routinely grown at 37 “C and 5% COz in a humid atmosphere, in Dulbecco’s modified Eagle’s medium (Gibco Laboratories), supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 pg/ml), and amphotericin B (2.5 pglml). For serum deprivation studies, cells were plated a t a density of 10,000 cells/cm*, allowed to grow to confluence, and thenshifted to a medium containing 0.5% fetal calf serum for 48 h to achieve quiescence. They were then changed into a medium with 15% fetal calf serum to induce synchronous entry into the cell cycle. Cycloheximide, when used, was added directly from a lOOOX aqueous solution stored at -20 “C, to a final concentration of 10 pg/ml. To obtain alarge number of mitotic cells, an exponentially growing culture was exposed to 0.4 pg/ml nocodazole (Sigma) for 8 to 10 h (22). The nonadherent cells were then collected by rocking and gentle squirting of the monolayer. The mitotic index was determined visually in these cells (23). The mitotic cells were washed three times with Earle’s balanced solution and thenseeded in normal medium, allowing them to proceed synchronously through the next cell cycle. Synchronization by a double thymidine block was performed according to Stein and Stein with minor modifications (24). Briefly, exponentially growing cells were treated with 2 mM thymidine for 14 h, then washed and cultured in fresh medium for 10 h, and exposed again to 2 mM thymidine for another 14 h to make then accumulate a t the GI-Sboundary. RNA Anulysis-Total RNA from cells was prepared by the acid/ guanidinium/phenol/chloroform method (25). For Northern blot analysis, aliquots of 15 pg of RNA solution were run in denaturalizing

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FIG. 1. PT-a mRNA expression in serum-deprivedcells stimulated to proliferate. A, cell sorter analysis of control (c) and serum-deprived cells 0, 14, 18, and 25 h after serum refeeding. Cells were harvested, and their DNA content was analyzed as described under “Materials and Methods.” B, Northern blot analysis and densitometric scanning of serum-deprived cells stimulated with 15% serum. RNA wasprepared at theindicated times after refeeding, and the levels of PT-a and histone H4 mRNAs were evaluated as described under “Materials and Methods.” Ethidium bromide-stained 28 S RNA is shown as a control for gel loading. 1.2% formaldehyde agarose gels (26). After electrophoresis at 3.5 V/ cm for 2 h, the gels were stained with ethidium bromide and transferred by electroblot to nylon membranes (Gene Screen, Du PontNew England Nuclear) following manufacturer’s instructions. RNA was fixed to the membrane by UV irradiation for 6 min (27). The cDNA probes employed for detection of PT-a (2), and histone H4 mRNAs (28) were labeled with ‘*P with a random primed kit (Boehringer Mannheim) following manufacturer’s instructions, to a specific activity of 1-2 X io9 cpm/pg. Prehybridization was carried out overnight at 42 “C in a solution containing 50% formamide, 0.2% polyvinyl pyrrolidone, 0.2% bovine serum albumin, 0.2% ficoll, 0.05 M Tris-HC1 (pH 7.5), 1 M NaCl, 0.1% sodium pyrophosphate, 0.1% SDS, 10% dextran sulfate, and 100 pg/ml denatured salmon sperm DNA. Hybridization was carried out for 24 h in the same solution with lo6 cpm/ml of column-purified labeled probe. Washing of the membranes for all probes was: twice in 2 X SSC (2 X SSC = 0.3 M NaCl, 0.03 M sodium citrate) at room temperature for 5 min; twice in 2 X SSC, 0.5% SDS at 65 “C for 30 min, and twice in 0.1 X SSC at room temperature for 30 min. Flow Cytometry-Sample processing was done according to Vindelov and Christensen (29) with minor modifications. DNA staining was done with propidium iodide (Calbiochem, Lot 910070). 1 ml of phosphate-buffered saline containing 5 pg of propidium iodide and 0.5% RNase Type I-A (Sigma) were added for at least 30 min and incubated in the dark at 4 “C. Finally, cells were filtered through a 35-pm nylon mesh to remove aggregates. Samples were then analyzed in a Coulter EPICS C fluorescence-activated cell sorter. Excitation was accomplished with an argon ion laser turned to 488 nm and operated at 300 milliwatts. Red fluorescence was measured with a long pass filter of 620 nm. Data were collected with a CYTOLOGY software program (Coulter). RESULTS

Gene expression of PT-a was investigated in serum-deprived NIH 3T3 fibroblasts that were stimulated to re-enter

0 CYCLOHEXlYlOE

FIG. 2. Effect of cycloheximide on PT-a mRNA expression after growthstimulation. Serum-deprived cells were treated with medium containing 15% fetal calf serum in the absence (control) or in the presence ( C H X )of 10 pg/ml cycloheximide. RNA was prepared at theindicated times post-treatment,and thelevels of PT-a, histone H4, and @-actinwere evaluated as described under “Materials and Methods.” Ethidium bromide-stained 28 S RNA is shown as a control ofgel loading. Densitometric analysis corresponds to PT-CY mRNA levels.

synchronously into thecell cycleby refeeding them with fetal calf serum-containing medium. To assess the synchronism obtained, cells were analyzed for their relative contents of DNA per cell by flow cytometry. More than 90% of serumdeprived cells hada Go-GI DNA content (Fig. lA). PT-a mRNA was induced 14 h after serum stimulation. HistoneH4 mRNA, that is known to appear duringthe GI to S transition (30) was induced at thesame time, placing PT-a elevation in the GI toS transition (Fig. 1B). We also wanted to study whetherPT-a mRNA was induced directly by serum, or itneeded the synthesis of one or several intermediate factors to occur. To address this issue, we induced quiescent cells with serum in the presence of 10 pg/ml cycloheximide,a concentrationthat inhibits protein synthesis in our cells approximately 95% (data notshown). Under these conditions, PT-a mRNA induction did not appear (Fig. 2). In the same experiment, &actin was superinduced, as it is expected from an early induced gene (31). To study PT-a mRNA cell cycle regulation, we used the mitotic shake off technique after nocodazole arrest in metaphase. As this method does not make cells quiescent, cells synchronized this way represent continuously cycling cells (23). To evaluate the synchronization obtained, we fixed and stained cells obtained by shake off and found them tobe 85% mitotic as scored visually. We also analyzed their DNA content by cell sorter analysis, and 90% had a 4n DNA content. Cells had apolyploidy of 5-10%. When we analyzed the DNA content of cells that had been reseeded, we found that 13 h after reseeding, most cells had an S phase content, and,at 20 h, they had a G,-M content, with some cells already in GI. On the contrary, 4 h after reseeding, there were apparently two populations of cells, one with 2n and the other with 4n

Prothymosin a mRNA Levels and Cellthe

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A. content of DNA (Fig. 3A). It has been described that when cell sorter analysis is performed in recently seeded cellssynchronized with the method we used, it registers nondetached daughter cells as a singlecell with doubleDNA content, although mitotic biochemical events have finished 30 min I A h after reseeding (22). To confirm that this was our case, we fixed and stained cells 2 h after reseeding. They were seen as E. pairs of cells with a reconstituted nuclear envelope (data not 0 2 4 6 8 10 HOURS shown). We concluded that cells had already divided and had entered G , as soon as they were reseeded. PT-alpha When we measured PT-a mRNAin these synchronized cells, we found its levels roughlyconstant throughout the cell HISTONE H 4 Om cycle (PT-a mRNA levelsas measured by scanning densitometry varied 520% between any two different hours in an individual experiment). On the contrary, the mRNA levelsof 28s histone H4, a well known cell cycle-regulated gene(30), were almost undetectable in mitosis and G,, had a dramatic increase during S phase (10- to 30-fold), and diminished again in G2 (Fig. 3B). Cells synchronized by the mitotic shake off method were very well synchronized during G , and S phases of the cell cycle, but their degree of synchrony fell to less than 60% 0 2 4 8 8 1 0 during G,. As it has been proposedthat PT-a exerts an action HOURS in GP(17), we were interested in studying its mRNA levelsat that moment. Thus, we synchronized our cells by a double PT-olpho 0 HISTONE H4 thymidine block inthe G, to S transition, so that they arrived FIG. 4. PT-a mRNA expression in cells synchronized by a at G2with a high degreeof synchrony. As can be seen in Fig. double thymidine block.A , cell sorter analysis of control cells (c) 4A, 6 h after therelease of the block more than 75% of cells and cells synchronized by double thymidine block 0,6,and 14 h after were in GI”, and synchronism was lost 14 h after block the release of the block. Cells wereharvested, and theirDNA content

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was analyzed as described under “Materialsand Methods.” B, Northern blot analysis and densitometric scanning of cells synchronized by double thymidine block. RNA was prepared at the indicated times and histone H4 after release of the block, and the levels of PT-LU mRNAs were evaluated as described under “Materialsand Methods.” Ethidium bromide-stained 28 S RNA is shown as a control for gel loading.

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release. PT-a mRNA did not vary significantly in any of the phases of the cell cycle (PT-a mRNA levels as measured by scanning densitometry varied 540% between any two different hours in an individual experiment). By contrast, histone H4 had a great elevation in S phase, as expected (Fig.4B). DISCUSSION

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FIG.3. PT-a mRNA expressionin cells synchronizedby mitotic shake off after nocodazole arrest. A , cell sorter analysis of control cells (c) and cells synchronized by mitotic shake off after nocodazole arrest 0, 4, 13, and 20 h after the shake off. Cells were harvested, and their DNA content was analyzed as described under “Materials and Methods.” B, Northern blot analysis and densitometric scanning of cells synchronized by mitotic shake off after nocodazole arrest. RNA wasprepared at theindicated times after reseeding, and the levels of PT-(Uand histone H4 mRNAs were evaluated as described under “Materialsand Methods.” Ethidium bromide-stained 28 S RNA is shown as a control for gel loading.

Serum-deprived NIH 3T3 cells didnot express, or expressed at very low levels, PT-CY mRNA, when these cells were stimulated to proliferate by serum refeeding, PT-a mRNA was induced; similar results were already published (3). The time course of PT-a mRNA induction after serum restoration also agrees with published data(3), and, by comparisonwith histone H4 mRNA induction, it seemed to occur in the GI to S transition (30). Our present data on PT-a mRNA behavior follows that of myc mRNA ( a ) myc levels increase 20-fold shortly after cells are stimulated to proliferate (32) and ( b ) levels of myc mRNA are invariant throughout the cell cycle (33). Therefore, our data are consistent with myc being an important stimulus of PT-a transcription (16),and, in agreementwith this, we found that protein synthesis was needed for PT-a induction. The synchronization of serum-deprivedNIH 3T3 by serum refeeding is a good model for studying the induction of cell division from Go cells, but cells progressively losetheir synchrony. Therefore, we usedcellssynchronized by mitotic shake off after nocodazole arrest to study PT-a mRNA levels in other phases of the cell cycle. Wefound that PT-a mRNA did not vary significantly in metaphase, G,, S, and even GP, although cells had a lower synchronization in that phase. PT-

Prothymosin a mRNA Levels and theCell Cycle mRNA was present at theend of the GI phase after serum refeeding and throughout the GIphase in cycling cells. Similar results were obtained with genes necessary for cellular proliferation (34). PT-a mRNA levelswere similar in nocodazole-arrested cells and in cells released from the arrest. Since nocodazolearrested cells are transcriptionally inactive, and nocodazole treatment lasted 10 h, the presence of PT-(UmRNA in these cells indirectly suggests that itis a stablemRNA, as previously suggested by Bishop’s group (16). Further confirmation to this supposition comes from the fact that 24 h were required for PT-a mRNA levels to be almost undetectable after serum deprivation (data not shown). The experiments with cells synchronized by both mitotic shake off and double thymidine block demonstrate that PTa mRNA levels exist in similar levels in all phases of cell cycle. On the other hand, histone H4 mRNA, a well known cell cycle-regulated gene (30), showed oscillating levels throughout the cell cycle. This led us to conclude that PT-a genes, ashappens with other genes necessary for cellular proliferation (34), are not regulated by the cell cycle. The behavior of PT-a mRNA described here is consistent with its proposed role in Gz. Nevertheless, if PT-a has an action in a specific phase of the cell cycle, as can be inferred from the experiments of Berger’s group (17), it should act in association with proteins specific of a particular phaseof the cell cycle or, alternatively, suffer cell cycle-regulated posttranslational modifications. Acknowkdgments-We are grateful to Prof. D. Schumperli for his gift of the recombinant plasmid containing the histone H4 probe. REFERENCES

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