cDNA for ProTa to determine which thymic cell populations are responsible for ProTa ..... mitotic activity, and a 4-fold increase in hairy cell leukemia, unknown.
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264 No. 15, Issue of May 25, pp. 8451-8454,1989 0 1989 by The American Societ; for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Communication 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 deBioquimica y Biologia Molecular, Facultad de Biologia, Uniuersidad de Santiago, Santiago deCompostela, Spain
esis thymosin a1 wouldbe secreted by the epithelium to modulate the immunological maturation of thymocytes during theirstayinthethymus (8). However, other works have shown that thymocytes do contain ProTa (10). Therefore, the elucidation of the intrathymic site of ProTa synthesis aswell 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 todetermine 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.
We isolated the cDNA for human prothymosin a (ProTa) from a human peripheral T-cell library using two synthetic oligonucleotides as probes. Hybridization studies with this cDNA showed that the ProTa mRNA is detectable in all the rat tissues studied but is most abundantin thymus and within thisgland mainly synthesized by thymocytes. In the T-cell lineage, its expression is higher in proliferative immature thymocytes than in pre- and post-thymic T lymphocytes. A quite similar pattern was obtained with the proliferation-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 ofProTa transcript is increased in lymphocytes from human patients with leukemias. Our findings indicate a role for ProTa linked to lymphocyte proliferation.
EXPERIMENTALPROCEDURES
Screening of the T-cell cDNA Library-The X g t l O library containing the human peripheral T-cell cDNAs was a gift from D. R. Littman (University of California, SanFrancisco) (11). The phages were screened by the method of Benton and Davis (12) with two synthetic oligonucleotide probes, whose sequences (5”GAGATCACCACwere CAAGGACCTG-3’ and 5’-GTCGTCCTCGTCGGTCTT-3’) based on the regions from theN and C termini of rat ProTaaccording 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 S E T (1 X = 0.15 M NaC1, 30 mM Tris-HC1, pH 8.0, 2 mM EDTA), 10 X Denhardt’ssolution,and 75 pg/mlEscherichia coli carrier DNA. Nucleotide Sequence Determination-The 850-, 1050-, and 1150base pair insertsof phages Xgt23, Xgt21, and Xgt31 selected from the library were subcloned intopUC8 vector (14). The recombinant plasmids were named pTC23, pTC21, and pTC31.Nucleotidesequences 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 theprocedure described by Poncz et al. (16). Thirty micrograms of double-stranded DNA from pTC31 were Prothymosin a is an acidic protein first isolated from rat linearized at the PstI site anddigested a t 30 “C with9 units of Ba131 thymus (1). It has been proposed to have a role in the in 600 mM NaCl, 12 mM CaC12,1 2 mM MgCl,, 20 mM Tris-HC1, pH regulation of cellular immunity based on several i n uiuo and 8.0, 1 mM EDTA. Aliquots were removed at various times and the of Ba131 digestion determined by electrophoresis in0.5 % agarose in uitro assays (2). Thus, ProTa’ is able to enhance resistance rate gels and 5% acrylamide gels. Aliquots containing deletions of desired to opportunistic infections in some animals (2) and appears size were pooled, phenol extracted, and precipitated with 2 volumes to restore deficient autologous and allogeneic mixed lympho- of ethanol. The DNA fragments were blunt-ended, EcoRI-digested, cyte responses in humans with active multiple sclerosis or and subcloned into M13mp8 cut with EcoRI and SmaI in order to of the remaining portion of the original systemic lupus erythematosus (3, 4). However, other studies maintain the same orientation Nucleotide sequence was determined have located ProTa in immune and nonimmune tissues ( 5 ) insert relative to the primer site. and have shown that its expression is induced upon stimula- by the dideoxy chain termination method (17). Cell Separation-Thymocytes were obtained by free diffusion from tion of cell growth (6). Therefore, the controversy over its rat thymus fragments. Cultured thymic stromal cells (fibroblasts and biological role still persists. epithelial cells) were from a confluent monolayer of the adherentcells Immunohistochemical studies on thymus with antibodies obtained from thymus explants essentially as described by Cohen et were separated by differential against thymosin a1 (7), a proteolytic derivative of ProTa al. (18).Bonemarrowpre-T-cells comprising the first 28 ProTa amino acid residues, have flotation in bovine serum albumin (Sigma) gradients (19). Thymolocated this peptide only in epithelial cells (8,9).Based on this cytes were separated according to their size into large and smallcells by centrifugation onto Percoll (Pharmacia LKB Biotechnology Inc.) immunolocation and certain activities found i n uitro, a hor- discontinuous density gradients (20). Peripheral lymphocytes were monal function for thymosin a1was proposed. In thishypoth- obtained on Ficoll (Pharmacia) density gradients (21) and spleen Ig’ and Ig- lymphocytes separated on antibody-coated plates (22). Large * This work was supported by Grant PR84-0627 from the Spanish thymocytes were selected by PNA (Sigma) agglutination (23). Comisibn Asesora Cientifica y TBcnica. The costs of publication of Northern Blot Analysis-Total RNA from tissues or cell populathis article were defrayed in part by the payment of page charges. tions from 3-month-old Wistar ratswas prepared by the acid guaniThis article must therefore be hereby marked“aduertisement”in dinium-phenol method (24). For Northern analysis, 15 pg of total accordance with 18 U.S.C. Section 1734 solely to indicate this fact. RNA or 1pg of poly(A)+ RNA (25)were separated ina 1.5% agaroseThe abbreviations used are: ProTa, prothymosin a; PBL, periph- formaldehyde gel (26) and transferred to nitrocellulose. Hybridization eral blood lymphocytes; PNA, peanut agglutinin; PNA’”, low PNA conditions were 50% formamide, 5 X SSPE (1X SSPE = 0.18 M NaC1, affinity thymocytes; PNAh’, high PNA affinity thymocytes; Ig+, im10 mM NaH2P04, pH 7.4, 1 mM EDTA), 10% dextran sulfate, 2 X munoglobulin positive; Ig-, immunoglobulin negative; PCNA, prolifDenhardt’s solution, 0.1% SDS, 100 pg/ml salmon testes DNA, and erating cell nuclear antigen; SDS, sodium dodecyl sulfate. 5 X lo6 cpm/ml nick-translated ProTcv cDNA at 42 “C for 16 h. Final ~
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ProTa mRNA in Tissues, Leukemias, and Lymphocytes
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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.
Location-Every rat tissue surveyed by Northern blot analysis contained a single RNA species of 1300 nucleotides that hybridized with the ProTacDNA probe (Fig. 2 A ) . The quantitation of the autoradiographs by densitometric scanning indicated that ProTa mRNA was most abundant in thymus, the organ where thymocytes are selected to yield mature Tcells. High levels were also detected in ovary (69% relative to RESULTS AND DISCUSSION thymus), kidney (66%),brain (44%), andheart(39%).In the amount was surprisingly high since Cloning of the Human T-cell ProTa cDNA-Based on the brainandheart, published sequence of rat ProTa, two oligonucleotides were previous studies were unable to detect this mRNA in those designed according to codon usage in eukaryotes (13) andused tissues (6), although in brain the peptide was identified (5). to screen a human T-cell cDNA library in hgtl0. From 40 x Large intestine (33%), lung (25%), cerebellum (21%), testis lo3 clones, we isolated the three positive clones Xgt21, Xgt23, (19%), small intestine(15%),and spleen (13%) presented and Xgt3l of 1050,850, and 1150 base pairs, respectively. The relative low quantities of ProTa mRNA. The lowest levels inserts from the three phage clones were liberated by EcoRI were in liver (6%) and striated muscle (5%). These datashow digestion and subcloned into the EcoRI site of the plasmid that the ProTa gene is expressed in all the tissues tested, pUC8 and termed pTC21, pTC23, and pTC31. Direct sequenc- even not immune-related, suggesting a general role for this ing, using the forward M13 primers, showed that the inserts protein. from the three clones contained an open reading frame which TOelucidate which thymic cell type is responsible for the encoded human ProTa. Thesequencing of the Ba131 deletion synthesis of ProTa, poly(A)+ RNA from thymocytes and mutants library of the pTC31 insert showed that this 1.1- cultured thymic stromal cells was probed with the ProTa kilobase pair cDNA contained the entire coding sequence of cDNA (Fig. 2B).Densitometric analysis of the autoradiograph ProTa (Fig. 1).The coding sequence of the pTC31 cDNA was indicates that the ProTa mRNA content is approximately 50found to be identical to the ProTa sequence reported by fold higher in thymocytes than in stromal cells. These results Goodall et al. (27), lacking the GAG codon in positions 295- argue against the hormonal hypothesis since (i) thesynthesis 297 of the fibroblast sequence published by Eschenfeldt and of ProTa and therefore its dervative thymosin a1 occurs Berger (6). The insert from pTC31 was used as the hybridi- mainly in thymocytes and (ii) RNA analysis shows a widezation probe in the ProTa expression studies. spread distribution of ProTa mRNA. Northern Blot Analysis of Rat Tissues andIntrathymic ProTa Gene Expression in Lymphoid Populations-The high amount of ProTa transcript in thymocytes led us to A analyze its levels in intra- and extrathymic lymphoid cells zso aoo -1ss 150 lop0 from different stages of maturation. We found this mRNA 0"UT *"UT more expressed in large thymocytes than in small nonmature I I EcoRl EcoRl (28) thymocytes (49% relative to large thymocytes), bone B marrow pre-T-cells (40%),and spleen Ig- lymphocytes (23%), Accl while virtually absent from spleen Ig+ lymphocytes (2%) (Fig. Ddel 3A). Large thymocytes, a heterogeneous population comprisHaelll ing both mature and immature cells (20), were fractionated Hinf I by binding to PNA intohigh (PNAhi) and low (PNA'O) affinity Hpa II thymocytes (23). PNAhithymocytes, immature cells undergoRsal 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 dataindicate 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 decreasing 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 sharetwo important features: the acidic character and the mRNA induction upon growth stimulation (6). The levels of the PCNA/cyclin mRNAwere FIG. 1. Restriction map and nucleotide sequence of ProTa cDNA. A, the structure of the cDNA including untranslated (UTi determined in the same filter, after complete washing and and coding (hatched area)regions. Arrows represent range and direc- extensive autoradiography, so that they were strictly compation of the sequencing read. B, the restriction map as determined rable to each other. The hybridization of the RNA from from the cDNA sequence. C, the nucleotide sequence of the ProTol thymocyte populations with the PCNA/cyclin probe is shown cDNA and the amino acid sequence deduced from it. The underlined in Fig. 3B,lower panel. The densitometric scanning of the regions correspond to thestretches used to design the oligonucleotide probes. The dashed line indicates the consensus polyadenylation autoradiograph showed an identical pattern for the PCNA/ cyclin mRNA. This finding would support a correlation besequence. I
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in Tissues, Leukemias, and Lymphocytes
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FIG.2. Distribution of ProTa mRNA in rat tissuesandintrathymic location. A, Northern analysis of total RNA (15 p g ) from 13 rat tissues (from left to right): striatedmuscle, large intestine, small intestine,ovary, cerebellum, brain, testis, spleen, liver, lung,thymus, heart,and kidney. Hybridization conditions were as described under "Experimental Procedures. " R, Northern analysis of poly(A)' RNA from thymic cells. Lane I , cultured stromal cells; lane 2, thymocytes. The experiment was performed as described above, exceptthat 1 pg of poly(A)+ RNAwas used.
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tween the proliferation state of the T-cells and the expression of the ProTagene. ProTa Gene Expression in Leukemic Lymphocytes-To test the possible relationship of ProTa gene expression with cell proliferation in uiuo, we decided to compare thelevels of ProT mRNA in lymphocytes obtained from humanleukemic blood with those in normal PBL using Northern blot analysis (Fig. 4). The densitometric scanning of the autoradiographshowed a &fold increase in acute lymphocytic leukemia, whose predominant cell type is an immaturelymphoid cell with a high mitotic activity, and a 4-fold increase in hairy cell leukemia,
leukemia (HCL), acute lymphocyticleukemia (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 autoradiographshown in the upper panel.
unknown. The data presented here show that ProTa gene expression changes during the maturationof 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 induction upon growth stimulation found by Eschenfeldt andBerger (6). In this view, the different levels of ProTa mRNA found in various tissues most likely reflect the presence of actively proliferating cells ratherthancontamination of lymphoid cells. Ourfindings reveala correlation between
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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. REFERENCES 1. Haritos, A.A., Goodall, G. J., and Horecker, B. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,1008-1011 2. Haritos, A. A,, Salvin, S. B., Blacher, R., Stein, S., and Horecker, B. L. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1050-1053 3. Reclos, G. J., Baxevanis, C. N., Sfagos, C., Papageorgiou, C., Tsokos, G. C., and Papamichail, M. (1987) Clin. Exp. Immunol. 70,336-344 4. Baxevanis, C. N., Reclos, G. J., Papamichail, M., and Tsokos, G. C. (1987) Immunopharmacol. Immunotoxicol. 9,429-440 5. Haritos, A. A., Tsolas, O., and Horecker, B. L. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,1391-1393 and Berger, S. L. (1986) Proc. Natl. Acad. 6. Eschenfeldt, W. H., Sci. U. S. A. 83,9403-9407 7. Goldstein, A.L.,Low, T. L. K.,McAdoo,M., McClure, J., Thurman, G. B., Rossio, J., Lai, C.-Y., Chang, D., Wang, S.-S., Harvey, C., Ramel, A. H., and Meienhofer, J. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 725-729 8. Dalakas, M. C., Engel, W . K., McClure, J. E., Goldstein, A.L., and Askanas, V. J. (1981) J.Neurol. Sci. 50, 239-247 9. Oates, K. K., Naylor, P. H., and Goldstein, A. L. (1987) Hybridoma 6,47-59 10. Freire, M., Rey-Mkndez, M., G6mez-Marquez, J., and Arias, P. (1985) Arch. Biochem. Biophys. 239,480-485 11. Littman, D. R., Thomas, Y., Maddon, P. J., Chess, L., and Axel, R. (1985) Cell 40,237-246
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