Communication Vol. 264 No. 15, Issue of May 25, pp. 8451-8454,1989 THE JOURNAL OF BIOLOGICAL CHEMISTRY

0 1989 by The American Societ; for Biochemistry and Molecular Biology, Inc. Printed in U. S. A.

The Exmession of Prothsmosin a. Gene in’T Lymphocytes and Leukemic Lymphoid Cells Is Tied to LymphocyteProliferation*

(Received for publication, November 21, 1988) Jaime Gomez-Marquez, Fernando Segade, Mercedes Dosil, Jose G. Pichel, Xose R. Bustelo, and Manuel Freire From the Departamento de Bioquimica y Biologia Molecular, Facultad de Biologia, Uniuersidad de Santiago, Santiago de Compostela, Spain

We isolated the cDNA for human prothymosin a (ProTa) from a human peripheral T-cell library using two synthetic oligonucleotides as probes. Hybridiza- tion studies with this cDNA showed that the ProTa mRNA is detectable in all the rat tissues studied but is most abundant in thymus and within this gland mainly synthesized by thymocytes. In the T-cell lineage, its expression is higher in proliferative immature thymo- cytes than in pre- and post-thymic T lymphocytes. A quite similar pattern was obtained with the prolifera- tion-related protein proliferating cell nuclear antigen/ cyclin. These data show that ProTa mRNA levels change with the maturation stage of T-cells. Moreover, the amount of ProTa transcript is increased in lympho- cytes from human patients with leukemias. Our find- ings indicate a role for ProTa linked to lymphocyte proliferation.

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Prothymosin a is an acidic protein first isolated from rat thymus (1). It has been proposed to have a role in the regulation of cellular immunity based on several i n uiuo and in uitro assays (2). Thus, ProTa’ is able to enhance resistance to opportunistic infections in some animals (2) and appears to restore deficient autologous and allogeneic mixed lympho- cyte responses in humans with active multiple sclerosis or systemic lupus erythematosus (3, 4). However, other studies have located ProTa in immune and nonimmune tissues ( 5 ) and have shown that its expression is induced upon stimula- tion of cell growth (6). Therefore, the controversy over its biological role still persists.

Immunohistochemical studies on thymus with antibodies against thymosin a1 (7) , a proteolytic derivative of ProTa comprising the first 28 ProTa amino acid residues, have located this peptide only in epithelial cells (8,9). Based on this immunolocation and certain activities found i n uitro, a hor- monal function for thymosin a1 was proposed. In this hypoth-

* This work was supported by Grant PR84-0627 from the Spanish Comisibn Asesora Cientifica y TBcnica. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: ProTa, prothymosin a; PBL, periph- eral blood lymphocytes; PNA, peanut agglutinin; PNA’”, low PNA affinity thymocytes; PNAh’, high PNA affinity thymocytes; Ig+, im- munoglobulin positive; Ig-, immunoglobulin negative; PCNA, prolif- erating cell nuclear antigen; SDS, sodium dodecyl sulfate.

esis thymosin a1 would be secreted by the epithelium to modulate the immunological maturation of thymocytes during their stay in the thymus (8). However, other works have shown that thymocytes do contain ProTa (10). Therefore, the elucidation of the intrathymic site of ProTa synthesis as well as its relation with the intrathymic maturation of T-cells in uiuo is important in order to clarify the intracellular function of ProTa.

For these purposes, we first isolated a human lymphocyte cDNA for ProTa to determine which thymic cell populations are responsible for ProTa synthesis. In addition, we analyze the levels of ProTa mRNA in lymphoid and nonlymphoid cells, including proliferative leukemic lymphocytes.

EXPERIMENTAL PROCEDURES

Screening of the T-cell cDNA Library-The X g t l O library contain- ing the human peripheral T-cell cDNAs was a gift from D. R. Littman (University of California, San Francisco) (11). The phages were screened by the method of Benton and Davis (12) with two synthetic oligonucleotide probes, whose sequences (5”GAGATCACCAC- CAAGGACCTG-3’ and 5’-GTCGTCCTCGTCGGTCTT-3’) were based on the regions from the N and C termini of rat ProTa according to published codon usage frequencies (13). The hybridization to the phage-containing nitrocellulose filters was carried out a t 37 “C for 16 h in 2 X SET (1 X = 0.15 M NaC1, 30 mM Tris-HC1, pH 8.0, 2 mM EDTA), 10 X Denhardt’s solution, and 75 pg/ml Escherichia coli carrier DNA.

Nucleotide Sequence Determination-The 850-, 1050-, and 1150- base pair inserts of phages Xgt23, Xgt21, and Xgt31 selected from the library were subcloned into pUC8 vector (14). The recombinant plasmids were named pTC23, pTC21, and pTC31. Nucleotide se- quences of inserts were determined by direct sequencing using the dideoxy chain termination method (15). To determine the complete nucleotide sequence of pTC31, a library of Ba131 deletion mutants was constructed according to the procedure described by Poncz et al. (16). Thirty micrograms of double-stranded DNA from pTC31 were linearized at the PstI site and digested a t 30 “C with 9 units of Ba131 in 600 mM NaCl, 12 mM CaC12, 1 2 mM MgCl,, 20 mM Tris-HC1, pH 8.0, 1 mM EDTA. Aliquots were removed at various times and the rate of Ba131 digestion determined by electrophoresis in 0.5 % agarose gels and 5% acrylamide gels. Aliquots containing deletions of desired size were pooled, phenol extracted, and precipitated with 2 volumes of ethanol. The DNA fragments were blunt-ended, EcoRI-digested, and subcloned into M13mp8 cut with EcoRI and SmaI in order to maintain the same orientation of the remaining portion of the original insert relative to the primer site. Nucleotide sequence was determined by the dideoxy chain termination method (17).

Cell Separation-Thymocytes were obtained by free diffusion from rat thymus fragments. Cultured thymic stromal cells (fibroblasts and epithelial cells) were from a confluent monolayer of the adherent cells obtained from thymus explants essentially as described by Cohen et al. (18). Bone marrow pre-T-cells were separated by differential flotation in bovine serum albumin (Sigma) gradients (19). Thymo- cytes were separated according to their size into large and small cells by centrifugation onto Percoll (Pharmacia LKB Biotechnology Inc.) discontinuous density gradients (20). Peripheral lymphocytes were obtained on Ficoll (Pharmacia) density gradients (21) and spleen Ig’ and Ig- lymphocytes separated on antibody-coated plates (22). Large thymocytes were selected by PNA (Sigma) agglutination (23).

Northern Blot Analysis-Total RNA from tissues or cell popula- tions from 3-month-old Wistar rats was prepared by the acid guani- dinium-phenol method (24). For Northern analysis, 15 pg of total RNA or 1 pg of poly(A)+ RNA (25) were separated in a 1.5% agarose- formaldehyde gel (26) and transferred to nitrocellulose. Hybridization conditions were 50% formamide, 5 X SSPE (1 X SSPE = 0.18 M NaC1, 10 mM NaH2P04, pH 7.4, 1 mM EDTA), 10% dextran sulfate, 2 X Denhardt’s solution, 0.1% SDS, 100 pg/ml salmon testes DNA, and 5 X lo6 cpm/ml nick-translated ProTcv cDNA at 42 “C for 16 h. Final

845 1

8452 ProTa mRNA in Tissues, Leukemias, and Lymphocytes

washes were at 65 "C in 0.1 X SSPE, 0.5% SDS. Calf thymus rRNA was used as a size standard.

To ensure that equal amounts of RNA were analyzed, the RNA concentration was determined by measuring the absorbance at 260 nm prior to gel loading. Using ethidium bromide staining we checked for the identical intensity shown by the ribosomal RNAs before and after transfer to the nitrocellulose filters.

RESULTS AND DISCUSSION

Cloning of the Human T-cell ProTa cDNA-Based on the published sequence of rat ProTa, two oligonucleotides were designed according to codon usage in eukaryotes (13) and used to screen a human T-cell cDNA library in hgtl0. From 40 x lo3 clones, we isolated the three positive clones Xgt21, Xgt23, and Xgt3l of 1050,850, and 1150 base pairs, respectively. The inserts from the three phage clones were liberated by EcoRI digestion and subcloned into the EcoRI site of the plasmid pUC8 and termed pTC21, pTC23, and pTC31. Direct sequenc- ing, using the forward M13 primers, showed that the inserts from the three clones contained an open reading frame which encoded human ProTa. The sequencing of the Ba131 deletion mutants library of the pTC31 insert showed that this 1.1- kilobase pair cDNA contained the entire coding sequence of ProTa (Fig. 1). The coding sequence of the pTC31 cDNA was found to be identical to the ProTa sequence reported by Goodall et al. (27), lacking the GAG codon in positions 295- 297 of the fibroblast sequence published by Eschenfeldt and Berger (6). The insert from pTC31 was used as the hybridi- zation probe in the ProTa expression studies.

Northern Blot Analysis of Rat Tissues and Intrathymic

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and coding (hatched area) regions. Arrows represent range and direc- tion of the sequencing read. B, the restriction map as determined from the cDNA sequence. C, the nucleotide sequence of the ProTol cDNA and the amino acid sequence deduced from it. The underlined regions correspond to the stretches used to design the oligonucleotide probes. The dashed line indicates the consensus polyadenylation sequence.

Location-Every rat tissue surveyed by Northern blot analysis contained a single RNA species of 1300 nucleotides that hybridized with the ProTa cDNA probe (Fig. 2 A ) . The quan- titation of the autoradiographs by densitometric scanning indicated that ProTa mRNA was most abundant in thymus, the organ where thymocytes are selected to yield mature T- cells. High levels were also detected in ovary (69% relative to thymus), kidney (66%), brain (44%), and heart (39%). In brain and heart, the amount was surprisingly high since previous studies were unable to detect this mRNA in those tissues (6), although in brain the peptide was identified (5). Large intestine (33%), lung (25%), cerebellum (21%), testis (19%), small intestine (15%), and spleen (13%) presented relative low quantities of ProTa mRNA. The lowest levels were in liver (6%) and striated muscle (5%). These data show that the ProTa gene is expressed in all the tissues tested, even not immune-related, suggesting a general role for this protein.

TO elucidate which thymic cell type is responsible for the synthesis of ProTa, poly(A)+ RNA from thymocytes and cultured thymic stromal cells was probed with the ProTa cDNA (Fig. 2B). Densitometric analysis of the autoradiograph indicates that the ProTa mRNA content is approximately 50- fold higher in thymocytes than in stromal cells. These results argue against the hormonal hypothesis since (i) the synthesis of ProTa and therefore its dervative thymosin a1 occurs mainly in thymocytes and (ii) RNA analysis shows a wide- spread distribution of ProTa mRNA.

ProTa Gene Expression in Lymphoid Populations-The high amount of ProTa transcript in thymocytes led us to analyze its levels in intra- and extrathymic lymphoid cells from different stages of maturation. We found this mRNA more expressed in large thymocytes than in small nonmature (28) thymocytes (49% relative to large thymocytes), bone marrow pre-T-cells (40%), and spleen Ig- lymphocytes (23%), while virtually absent from spleen Ig+ lymphocytes (2%) (Fig. 3A). Large thymocytes, a heterogeneous population compris- ing both mature and immature cells (20), were fractionated by binding to PNA into high (PNAhi) and low (PNA'O) affinity thymocytes (23). PNAhi thymocytes, immature cells undergo- ing continuous divisions (28), contained 3-fold more ProTa mRNA than postmitotic mature thymocytes (PNA'O) (Fig. 3B, upper panel). Normal PBL, which circulate in a resting state (29), showed low levels of ProTa mRNA transcription (Fig. 3A). These data indicate a differential transcriptional activity of the ProTa gene during the maturation of T-cells in vivo, increasing from pre-T-cells to immature thymocytes and de- creasing in mature thymocytes, spleen Ig- lymphocytes, and circulating lymphocytes.

The hybridization data in thymocyte populations seemed to indicate that the ProTa mRNA expression is related to the proliferation state of the thymocytes. The pattern found in the thymocyte subpopulations was compared with the levels of the mRNA of PCNA/cyclin, an acidic nuclear protein (30) whose synthesis is correlated with the proliferation state of the cells (31). Both proteins share two important features: the acidic character and the mRNA induction upon growth stim- ulation (6). The levels of the PCNA/cyclin mRNA were determined in the same filter, after complete washing and extensive autoradiography, so that they were strictly compa- rable to each other. The hybridization of the RNA from thymocyte populations with the PCNA/cyclin probe is shown in Fig. 3B, lower panel. The densitometric scanning of the autoradiograph showed an identical pattern for the PCNA/ cyclin mRNA. This finding would support a correlation be-

ProTa mRNA in Tissues, Leukemias, and Lymphocytes 8453

FIG. 2. Distribution of ProTa mRNA in rat tissues and intra- thymic location. A, Northern analysis of total RNA (15 pg) from 13 rat tissues (from left to right): striated muscle, large intestine, small intestine, ovary, cerebel- lum, brain, testis, spleen, liver, lung, thy- mus, heart, and kidney. Hybridization conditions were as described under "Ex- perimental Procedures. " R, Northern analysis of poly(A)' RNA from thymic cells. Lane I , cultured stromal cells; lane 2, thymocytes. The experiment was per- formed as described above, except that 1 pg of poly(A)+ RNA was used.

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FIG. 4. ProTa mRNA levels in human leukemic peripheral blood lymphocytes. Upper panel, from left to right: hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and normal PBL. RNA procedures were as in Fig. 2. Lower panel represents the quantitation of ProTa gene expression evaluated by densitometric scanning of the autoradiograph shown in the upper panel.

tween the proliferation state of the T-cells and the expression of the ProTa gene.

ProTa Gene Expression in Leukemic Lymphocytes-To test the possible relationship of ProTa gene expression with cell proliferation in uiuo, we decided to compare the levels of ProT mRNA in lymphocytes obtained from human leukemic blood with those in normal PBL using Northern blot analysis (Fig. 4). The densitometric scanning of the autoradiograph showed a &fold increase in acute lymphocytic leukemia, whose pre- dominant cell type is an immature lymphoid cell with a high mitotic activity, and a 4-fold increase in hairy cell leukemia,

unknown. The data presented here show that ProTa gene expression changes during the maturation of T-lymphocytes. The increased levels in immature proliferating thymocytes and in human leukemias point to a function related to a lymphocyte proliferation. This hypothesis is strengthened by the identical expression pattern of the proliferation-related PCNA/cyclin in thymocyte subpopulations and by the induc- tion upon growth stimulation found by Eschenfeldt and Ber- ger (6). In this view, the different levels of ProTa mRNA found in various tissues most likely reflect the presence of actively proliferating cells rather than contamination of lymphoid cells. Our findings reveal a correlation between

a454 ProTa mRNA in Tissues, Leukemias, and Lymphocytes

ProTa mRNA levels and the T-lymphocyte maturation and perhaps a more general function in cell proliferation.

Acknowledgments-We thank D. R. Littman for the gift of the lymphocyte cDNA library, R. Bravo for the S14 cyclin cDNA, J. M. Castro Freire for the thymic stromal RNA, and the Hospital General de Galicia for the leukemic blood samples.

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