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Is thymosin α1 a thymic hormone?
CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY Vol. 65, No. 3, December, pp. 195-200, 1992
SHORT ANALYTICAL REVIEW Is Thymosin (x1 a Thymic Hormone? PAUL SZABO AND MARC E. WEKSLER Division of Geriatrics and Gerontology, Cornell University Medical College, New York, New York 10021 INTRODUCTION
The thymus gland is the principal site of differentiation of bone marrow-derived precursors of T lymphocytes. Based largely on its histological appearance, the thymus gland had also been thought to be an endocrine organ. In an attempt to prepare compounds with hormonal properties, aqueous extracts of bovine thymic glands were prepared. A complex mixture of peptides ranging in molecular weight from 1 to 14 kDa was designated thymosin fraction V. Despite the reported capacity of this fraction and some of its component peptides to enhance in vitro or in viuo assays of immune function and enhance immune function in patients with immunodeficiency disease (1, 21, the immunoregulatory function of thymic peptides remains controversial. THYMOSIN
a1
The first peptide in thymosin fraction V to be purified to homogeneity was a highly acidic, 28 amino acid peptide named thymosin o1 (3,4). Many of the biological properties of thymosin fraction V were possessed by this polypeptide. Subsequently, other peptides have also been purified from thymosin fraction V and some of these have also been reported to have biologic activity (5-7). In this review, the origin and biological function of thymosin c+ the best characterized of the peptides in thymosin fraction V, will be considered. As the amino acid sequences of the acidic peptides making up thymosin fraction V were determined, it became clear that a number of peptides shared the same amino terminus, an acetylated serine residue, and had identical amino terminal sequences although they differed in size (8). One peptide was a 24 amino acid polypeptide, thymosin (des 2&28), consisting of the first 24 amino terminal amino acids of thymosin (pi. Another peptide (thymosin ali) was seven amino acids shorter than thymosin al. Larger polypeptides were also isolated which had the 28 amino acids of thymosin 0~~at their amino terminus. This raised the possibility that all of these peptides were derived from a common protein by nonspecific
proteolytic degradation rather than specific cleavage of a precursor protein. This conjecture was supported by the report that intact poly(A) + mRNA isolated from the rat thymus when added to a cell-free protein translation system produced a 16,000-Da product which reacted with an antibody specific for thymosin 0~~(9). The most compelling evidence in support of the origin of many of these peptides from a larger precursor came from the analysis of extracts of fresh rat thymus glands prepared under conditions that inhibited proteolysis. Under such conditions, a 113 amino acid polypeptide reactive with antibodies to thymosin (pi and containing at its amino terminus the 28 amino acid sequence of thymosin oi, but none of the small peptides that shared sequence with thymosin oi, was found (10). Furthermore, purified prothymosin was reported to exhibit many of the biological and immunological activities associated with thymosin 01~including protection against opportunistic infections (11). This purification procedure also yielded a second structurally related, highly acidic polypeptide from rat thymus glands, designated parathymosin (12). This protein inhibited some of the biological activities of prothymosin (11). Thymosin 0~~makes up less than 10% of the total immunoreactive peptide in either serum or red blood cells in uiuo and it has been suggested that this low level is actually the result of partial degradation of prothymosin OLand its leakage from leukocytes (13). In contrast, intact prothymosin cx is extremely abundant in circulating cells (50-70 pmol/ml) and appears to be concentrated in nucleated white blood cells including both polymorphonuclear and mononuclear leukocytes (13). The detection of prothymosin 01 and the failure to detect appreciable levels of thymosin o1 in human blood suggested that thymosin (rl might not actually be a circulating hormone but rather a proteolytic breakdown product of prothymosin (Y. Subsequent studies suggested that prothymosin (Yis not a protein limited to the thymus gland or even a protein limited to cells of immunological origin. Prothymosin (Y was abundant in all tissue examined in195 0090-1229/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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eluding kidney, liver, and brain (14). Prothymosin (Yis not thymic dependent as it was detected in the tissues of athymic animals as well as in old animals which lack a functional thymus gland. Thus, prothymosin (Yis a protein found in all cell types examined and is not limited to cells of the immune system. THE
STRUCTURE
AND
FUNCTION
OF
WEKSLER
a
b
PROTHYMOSIN
Additional evidence against thymosin oi being a hormone or that prothymosin (Yis a precursor to a hormone comes from sequence analysis of prothymosin (Y cDNA clones (15). There are no additional amino terminal amino acids encoded in the prothymosin (Y mRNA 5’ to the amino terminal acetylated serine that might function as secretory leader sequences. Only the initiator methionine residue is clipped off prior to acetylation of the serine residue (15). While alternative mechanisms for secretion of proteins have been reported (161, such secreted proteins are distinctly unusual. Prothymosin (Y mRNA is also associated with free, cytoplasmic ribosomes, not with rough endoplasmic reticulum-bound ribosomes (17) which would be expected of proteins that are secreted. Prothymosin cxis not only found in a wide variety of tissue types, including thymus, spleen, liver, kidney, brain and muscle, but its mRNA is also detectable in these tissues suggesting that de nouo synthesis of prothymosin (Y occurs in all these cell types (18). These findings suggested that prothymosin (Y may have a more general cellular function and may be required in many different cells and tissues. Proliferating thymocytes express relatively high levels of prothymosin (YmRNA (19). The elevated levels of prothymosin o mRNA seen in early stages of thymocyte development decrease as these cells mature and cease proliferation (19). Mature, circulating T cells do not express high levels of prothymosin (YmRNA under normal conditions (19, 20); however, when T cells are stimulated by mitogen to proliferate, the steady state level of prothymosin (YmRNA increases (20, 21). During the transition from the nondividing GO cells to proliferating cells, the level of prothymosin (Y mRNA increases four- to sixfold and peaks at mid-G1 to the S phase of the cell cycle. This is seen for both T and B lymphocytes (21). As shown in Fig. 1, when T cells, cultured with mitogen, are purified and separated into nonactivated and activated cells, the ratio of prothymosin (YmRNA in activated cells as compared to nonactivated cells is 20 to 1, suggesting that the activity of this protein is expressed at much higher levels in dividing cells. Transformed and proliferating leukemic cells also show increased levels of prothymosin (Y mRNA (19). Prothymosin 01mRNA expression in other cell types including fibroblasts also correlates with their proliferation. Serum-starved, nondividing NIH3T3 cells ex-
< ProT
FIG. 1. Prothymosin u expression in activated T lymphocytes. Shown is a Northern blot of total RNA from PHA-activated and quiescent human T-lymphocytes separated by Percoll gradient centrifugation. A Northern blot of RNA from fractions consisting of activated (a) and quiescent cb) cells was probed with 32P-labeled prothymosin cDNA. The level of prothymosin-specific mRNA in activated cells is >20-fold higher than that seen in the quiescent T cells.
press relatively low levels of prothymosin (Y mRNA. When these cells are induced to proliferate by the addition of serum, the level of prothymosin 01 mRNA increases significantly (22). Similarly, a rapid increase in prothymosin (Yexpression as measured by levels of thymosin immunoreactive peptide is seen in small intestinal crypt cells induced to proliferate by serum stimulation (23). Finally, prothymosin (Yn-RNA expression has also been observed in proliferating hepatocytes in a regenerating rat liver model system (20). As would be expected for a protein required for cell proliferation, prothymosin (Y mRNA expression is reduced in cells which cease dividing. Proliferating murine erythroleukemia cells (MELC) express high levels of prothymosin (YmRNA cultures. These cultures approach saturation density and cease proliferation and the level of prothymosin (YmRNA decreases by nearly two orders of magnitude (P. Szabo, unpublished observations). Similarly, the level of prothymosin 01 mRNA is reduced when HL60 human erythroleukemia leukemia cells are induced to terminally differentiate (24). Thus, the level of prothymosin (Y mRNA reaches negligible levels 1 hr after the exposure of HL60 cells to DMSO, which leads to their differentiation into neutrophils. Exposure of HL60 cells to tetradescanoyl phorbolacetate, TPA, results in a similar loss of prothymosin (YmRNA between 6 and 24 hr after this induction to differentiate into macrophages. The decline in prothymosin (Y mRNA occurs when MELC express their cell-specific differentiation products, e.g., globin. In the case of MELC, which also undergo terminal cell division, there is diminished expression of prothymosin (Y mRNA initially after the addition of the differentiation inducer (e.g., HMl3A), This initial decrease, which is like that seen for HL-60 cells, is followed by a secondary peak of expression at
IS THYMOSIN
a1 A THYMIC
36 to 48 hr after initiation of differentiation with HMBA. The time of the secondary peak varies with cell density and the inducer used. Another protein whose level is modulated by differentiation in these cell types and is associated with proliferation is the c-myc protein. The c-myc gene product, a progression factor, is a nuclear DNA-binding protein which functions as a transacting regulator presumably upregulating genes involved in cellular proliferation (see review (2.5)). Inhibition of the synthesis of c-myc protein prevents the replication of DNA and thereby blocks cellular proliferation. In an effort to determine what genes are specifically regulated by the c-myc protein, an estrogeninducible, recombinant construct containing the c-myc gene was transfected into rat fibroblasts (26). Estrogen induced transfected cells in the stationary phase to enter the cell cycle. Subtractive DNA libraries made from induced cells were screened for sequences that were induced by the expression of the c-myc protein. Initially, only a single clone was obtained which was sequenced and found to be the prothymosin CYgene. Thus, c-myc protein induces prothymosin 01 gene transcription. A potential binding site for c-myc has been detected 1200 base pairs 5’ to the transcriptional start site (P. Szabo, unpublished observations). The expression of c-myc in T cells induced to proliferate occurs early in Gl (27), preceding the appearance of prothymosin (YmRNA in mid to late G1(20,21). The addition of inducers of differentiation to MELC and HL60 cells leads to a fall in c-myc mRNA levels prior to the decrease in prothymosin OLmRNA. Furthermore, in MEL cells, a secondary peak of c-myc expression precedes the secondary peak of prothymosin (Ygene expression. These results are consistent with the direct activation of the prothymosin cx gene by the c-myc gene product. Thus, prothymosin CYappears to play a role in the normal progression of cells through the cell cycle and may, in fact, be required for the replication of DNA. Recently, the addition of antisense oligomers of prothymosin cx mRNA to synchronized human myeloma cells was found to delay cell division (28). The delay, without complete inhibition of cell cycle progression, is due to the instability of the antisense oligomers.
HORMONE?
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ies are not of high affinity for native prothymosin o, these results may not accurately reflect the distribution of this protein. Direct analysis of the distribution of prothymosin cx in cells was originally done by the injection of labeled prothymosin OLprotein into Xenopus Zueuis oocytes (31). The results of these studies demonstrated that prothymosin CYis rapidly accumulated in the germinal vesicle within 24 hr of injection (31). The related protein, parathymosin, which contains a similar nuclear target peptide, is also seen to accumulate in the germinal vesicle (32). To confirm that prothymosin OLtargets the nucleus, recombinant DNA constructs containing the prothymosin (Y cDNA sequence linked to the human growth hormone (hGH) gene were transfected into HeLa cells (33). The constructs were under the control of the SV40 (large T) promoter and were constitutively expressed in the transfected HeLa cells. The fusion protein derived from this construct could be identified specifically by antibodies to hGH by indirect immunofluorescence. Cells with constructs containing the entire hGH cDNA showed fluorescence in the perinuclear secretory (Golgi) apparatus as expected for a secretory protein (Fig. 2). Cells with the hGH cDNA constructs that lacked the sequences coding for the leader peptide showed diffuse fluorescence throughout the cytoplasm. Cells with the constructs that had the entire prothymosin a or parathymosin sequence flanking the leaderless hGH sequence showed fluorescence concentrated in the nucleus. Thus, both the prothymosin (Y and parathymosin polypeptides have nuclear targeting sequences and the fusion protein is transported to the nucleus. Similar studies using different recombinant constructs confirmed that the prothymosin (Y polypeptide is transported to the nucleus (34). Cells transfected with constructs which lacked the carboxyl terminus, which contained the consensus nuclear targeting sequence, did not transport the protein to the nucleus (34).
The peptide sequence of prothymosin CLis not homologous with any known protein; however, its central, highly acidic stretch of amino acids does show some homology to high mobility group 1 protein (20). This protein is involved in the release of histones from chromatin and it has been proposed that prothymosin (Y PROTHYMOSIN IS A NUCLEAR PROTEIN plays a similar role in the nucleus (20). Since the level 01 mRNA expression in some cells is When the protein sequence of prothymosin (Y was of prothymosin obtained a short stretch of amino acids (KKQKK) near elevated in Gl prior to cell entry into the S phase, it is the carboxyl terminus was found which resembled se- possible that prothymosin cx is required to open chroquences found in proteins that are transported to the matin structure in order to enable DNA to be replinucleus (29). Such a pentapeptide sequence is found in cated. However, the fact that low levels of prothymosin all nuclear targeting proteins examined to date and is CYmRNA are present throughout the cell cycle suggests thought to direct the transport of such proteins to the that this molecule may be also required for other nuclear processes, e.g., an enhancer of RNA transcripnucleus. Direct analysis of the distribution of prothy(Ymay be a secondary transcription mosin (Yin liver cells using antibodies for thymosin (Y~ tion. Prothymosin have identified this protein in both the cytoplasm and factor acting on a limited number of genes needed for the nucleus of cells (30). However, since these antibodproliferation (28).
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SZABO AND WEKSLER
Isv4oIss
1
hGH
I s
3
Iswl
hGH 13
3
IswI
hGH
1
hPAR4
1
FIG. 2. Nuclear targeting of prothymosin and parathymosin. Recombinant plasmid constructs were prepared to determine the intracellular localization of the thymosins. The reporter gene used in these constructs was human growth hormone cDNA under control of the SV40 large T promoter. The growth hormone product of this clone could be specifically detected using indirect immunofluorescence. The first panel shows this initial construct and a photomicrograph of HeLa cells transfected with this plasmid; fluorescence from the newly synthesized growth hormone is concentrated in the Golgi bodies as expected for a secreted peptide hormone. The second panel shows the results using a second construct in which the secretory signal sequence (ss) has been deleted, as expected, the fluorescence is spread throughout the cytoplasm of the transfected cells. The final panel shows a construct in which parathymosin cDNA has been cloned in proper reading from next to the growth hormone sequence; the fluorescence is found only in the nucleus, suggesting direct nuclear targeting of the parathymosin sequence. The results with constructs bearing prothymosin a, rather than parathymosin cDNA, are identical.
PROTHYMOSIN
GENE
The gene coding for prothymosin o. in humans has been mapped to the long arm of chromosome two (35). It’s structure consists of five exons within about 6.8 kb. The amino acid coding sequences are found in exons l-4; the fifth exon comprises 3’ untranslated sequence. These exons, some of the introns, and the 5’ flanking region have been sequenced (17,35). Numerous, potential &-acting regulatory elements have been identified in the 5’ flanking region (35) of the sequence CACGTG, and these include an E box element located about 1200 base pair 5’ to the start of transcription and known to be recognized by the c-myc:max complex. This later complex has been reported to be the functional transacting c-myc regulatory unit (36). This cisacting regulatory sequence is located about 1.2 kb upstream from the transcriptional start site; binding of the c-myc-max complex at this site may result in direct activation of the prothymosin a gene. In addition to this functional prothymosin a1 gene, there exist at least five prothymosin a-related pseudogenes dispersed in the human genome. These have been partially sequenced and appear to be cDNA pseudogenes (17). The gene coding for prothymosin CYis highly conserved; human cDNA probes crosshybridize to genomic DNA from mice, rats, chickens, fish, and fruit flies, (see Fig. 3). The protein itself has been detected in a wide
variety of species including yeast (37). In the case of murine and rat DNA and to a lesser extent chicken and fish DNA, multiple genomic sites with homology to prothymosin cxare detected by the human probe. It is not clear whether these represent multiple genes in these species or whether these species also contain pseudogenes. In Drosophila, the human prothymosin a cDNA probe appears to detect a unique gene. The evolutionary conservation of sequences for prothymosin a suggests that the function carried out by this protein is of major importance since the nucleotide sequence has been maintained for several hundred million years. CONCLUSION
When first described, the thymosins were thought of as modulators acting as hormones that primarily promoted the proliferation and differentiation of T lymphocytes. It has now become apparent that these compounds, even if they exhibit some immune activities, play a very different biological role. Prothymosin CY,the most well-studied thymosin and the precursor to thymosin ai, has a wide cellular distribution and is not restricted to cells of the immune system. Although its actual role is not known, prothymosin a appears to act primarily in the nucleus of cells and appears to be involved in cellular proliferation. Tran-
IS THYMOSIN
a
b
c
ai A THYMIC
d 5.
23.19.4-
6.
7. 8.
FIG. 3. Evolutionary conservation of the prothymosin Q gene shown is a Southern blot of EcoRI-digested DNA from mouse (lane a), rat (lane b), chicken (lane cl, and Drosophila melunogoster (lane d) annealed with human prothymosin alpha cDNA probe. Hybridization to numerous bands is seen for the two mammalian species presumably due to the existence of multiple pseudogenes. There is less intense hybridization and fewer bands seen for chicken and Drosophila DNA; the relatively dark bands seen in the Drosophila DNA compared to chicken DNA is the result of a fivefold greater DNA in terms of genome equivalents rather than greater homology to the human cDNA probe.
9.
10. 11.
12.
scription of prothymosin (Y may be directly controlled by the c-myc protein, a progression factor whose expression is required for DNA synthesis and cell division. The precise role of prothymosin CYas the cellular proliferation process is currently under intense investigation. ACKNOWLEDGMENTS This work was supported in part by the National Institute on Aging Program Project Grant (AG 00541). We acknowledge the major contributions of Dr. Bernard Horecker, both to our understanding of the thymosins and for his encouragement to enter this field. We thank Drs. L. Graeve, E. Rodriguez-Boulon, M. Clinton, C. Panneerselvam, M. Smith, A. Silverman, M. Mutchnick, and Mr. E. Whittington for information contributing to this review. We are grateful for the secretarial support of Liz Dugan.
13. 14.
15. 16.
17.
REFERENCES Low, T. L. K., and Goldstein, A. L., Thymosins: Isolation, structural studies and biological activities. In “Thymic Hormones and Lymphokines” (A. L. Goldstein, Ed.), Plenum Press, New York, 1984. Goodall, G. J., and Horecker, B. L., “Molecular biology of the Thymosins in Immune Regulation by Characterized Polypeptides,” pp. 283-292, A. R. Liss, New York, 1987. 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., Thymosin alpha-l: Isolation and sequence analysis of an immunologically active thymic polypeptide. Proc. Natl. Acad. Sci. USA 74, 725-729, 1977. Low, T. L. K., Thurman, G. B., Chincarini, C., McClure, J. E., Marshall, G. D., Hu, S-K., and Goldstein, A. L., “Current status
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of thymosin research: Evidence for the existence of a family of thymic factors that control T-cell maturation”. Ann N.Y. Acud. Sci. 332, 33-48, 1979. Schulof, R. S., and Goldstein, A. L., Thymosins and other thymic hormones. In “The Lymphokines: Biochemistry and Biological Activity” (J. W. Hadden and W. E. Stewart, Eds.), p. 379, Humana Press, Clifton, NJ, 1981. Low, T. L. K., Hu, S-K., and Goldstein, A. L., Complete amino acid sequence of bovine thymosin B4: a thymic hormone that induces terminal deoxynucleotidyl transferase activity in thymocyte populations. Proc. Nutl. Acad. Sci. USA 78, 1162-1166, 1981. Erikson-Viitaner, S., Ruggieri, S., Natalini, P., and Horecker, B. L., Thymosin B,,, a new analog of thymosin B, in mammalian tissues. Arch B&hem. Biophys. 225, 407-413, 1983. Cardarella, J., Goodall, G. J., Felix, A. M., Heimer, E. P., Salvin, S. B., and Horecker, B. L., Thymosin alpha 2, a peptide related to Thymosin alpha 1 isolated from calf thymosin fraction 5. Proc. Natl. Acad. Sci. USA 80, 7424, 1983. Freire, M., Hannappel, E., Rey, M., Freire, J. M., Kido, H., and Horecker, B. L., Purification of thymus mRNA coding for a 16,000-dalton polypeptide containing the thymosin alpha-l sequence. Proc. Natl. Acad. Sci. USA 78, 192-195, 1981. Haritos, A. A., Blacker, R., Stein, S., Cardarella, J., and Horecker, B. L., Primary structure of rat thymus prothymosin alpha. Proc Natl. Acad. Sci. USA 82, 343-346, 1985. Pan, L-X., Haritos, A. A., Wideman, J., Komiyama, T., Chang, M., Stein, S., Salvin, S. B., and Horecker, B. L., Human prothymosin alpha: Amino acid sequencing and immunologic properties. Arch. Biochem. Biophys. 250, 197-201, 1986. Haritos, A. A., Salvin, S. B., Blacker, R., Stern, S., and Horecker, B. L., Parathymosin alpha: A peptide from rat tissues with structural homology to prothymosin. Proc. Natl. Acud. Sci. USA 82, 1050-1053, 1985. Panneerselvan, C., Haritos, A. A., Cardarella, J., and Horecker, B. L., Prothymosin alpha in human blood. Proc. Natl. Acud. Sci. USA 84, 4465-4469, 1987. Haritos, A. A., Tsolas, O., and Horecker, B. L., Distribution of prothymosin alpha in rat tissues. Proc. Natl. Sci. USA 81, 13911393, 1984. Goodall, G. J., Dominquez, F., and Horecker, B. L., Molecular cloning of cDNA for human prothymosin alpha. Proc. N&Z. Acad. Sci. USA 83, 8926-8928, 1986. Matsushima, K., Taguchi, M., Kovacs, E. J., Young, H. A., and Oppenheim, J. J., Intracellular localization of human monocyte associated interleukin 1 (IL 1) activity and release of biologically active IL 1 from monocytes by trypsin and plasmin. J. Immunol. 136, 2883-2891, 1986. Eschenfeldt, W. H., Monrow, R. E., and Bergen, S. L., Isolation and partial sequencing of the human prothymosin alpha gene family: Evidence against export of the gene products. J. Biol. Chem. 264, 75467555,1989. Clinton, M., Frangou-Lazaridis, M., Panneerselvan, C., and Horecker, B. L., Prothymosin alpha and parathymosin: mRNA and polypeptide levels in rodent tissues. Arch. ofBiochem. Biophys. 269, 256-263, 1989. Gomez-Marquez, J., Segade, F., Dosil, M., Pichel, J. S., Bustelo, X. R., and Freire, M., The expression of prothymosin alpha gene in T lymphocytes and leukemic lymphoid cells is tied to lymphocyte proliferation. J. Biol. Chem. 264, 8451-8454, 1989. Bustelo, X. R., Otero, A., Gomez-Marquez, J., and Freire, M., Expression of the rat prothymosin alpha gene during T-lymphocyte proliferation and liver regeneration. J. Biol. Chem. 266, 1443-1447,199l. Szabo, P., Ehleiter, D., Whittington, E., and Weksler, M., Pro-
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SZABO thymosin alpha T lymhocytes. 1992.
expresion
occurs
during
Gl in proliferating
AND B or
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22. Eschenfeldt, W. H., and Berger, S. L., The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: Evidence using a cloned cDNA. Proc. Natl. Acad. Sci. USA 83, 9403-9407, 1986. 23. Conteas, C. N., Mutchnick, M. G., Palmer, K. C., Weller, F. E., Luk, G. D., Naylor, P. H., Erdes, M. R., Goldstein, A. L., Panneerselvan, C., and Horecker, B. L., Cellular levels of thymosin immunoreactivepeptides are linked to proliferative events: Evidence for a nuclear site of action. Proc. Natl. Acad. Sci. USA 87, 3269-3273, 1990. 24. Smith, M. R., Silverman, A., Szabo, P., Kohler, W., Nath, R., and Mutchnick, M. G. Prothymosin alpha gene expression is downregulated in differentiating HL-60. Submitted for publication. 25. Luscher, B., and Eisenman, R. N., New light on Myc and Myb: Part 1. Myc. Genes Dev. 4, 2025-2035, 1991. 26. Eilers, M., Schiron, tivates transcription 133-144,1991.
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27. Gamble, D., Schwab, R., Weksler, M.E., and Szabo, P., The ageassociated in vitro proliferative defect(s) in human T-lymphocytes precedes c-myc gene activation. J. Immunol. 144, 35693573,199o. 28. Sburlati, A. R., Manrow, R. E., and Berger, S. L. “Prothymosin alpha antisense oligomers inhibit myeloma cell division”. Proc. Natl. Acad. Sci. USA 88, 253-257, 1991. 29. Gomez-Marquez, J., and Segade, F., Prothymosin alpha is a nuclear protein. FEBS Lett. 226, 217-219, 1988. Received
June
25, 1992;
accepted
July
8, 1992
WEKSLER Komiyama, T., Pan, L-X., Haritos, A. A., Wideman, J. W., Pan, Y-CE., Chang, M., Rogers, I., and Horecker, B. L., The primary structure of rat parathymosin. Proc. Natl. Acad. Sci. USA 83. 1242-1245,1986. 30. Conteas, C., Su, Y-L., 3. A1151, 1989
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USA 88, 6608, 1991. 34. Manrow, R. E., Sburlati, A. R., Hanover, J. A., and Berger, S. L., Nuclear targeting of prothymosin alpha. J. Biol. Chem. 206. 3916. 1991. 35. Szabo, P., Panneerselvam, C.. Clinton, M., Frangou-Lazaridis. M., Weksler, D., Whittington, E., Macera, M. J., Grzeschik. K-H., Selvaknmar, A., and Horecker, B. L., Localization of the prothymosin alpha gene to chromosome 2 in humans and organization of its promoter region. Submitted for publication. 36. Blackwood, G. C., and Eisenman, R. N., Max: A helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with myc. Science 251, 1211-1217, 1991. 37. Makarova, YR, Grebenshikov, N., Egorov, C., Vartapetian, A,. and Bogdonov, A., Prothymosin alpha is an evolutionary conserved protein covalently linked to a small RNA. FEBS Lett. 257. 247-250. 1989.
SHORT ANALYTICAL REVIEW Is Thymosin (x1 a Thymic Hormone? PAUL SZABO AND MARC E. WEKSLER Division of Geriatrics and Gerontology, Cornell University Medical College, New York, New York 10021 INTRODUCTION
The thymus gland is the principal site of differentiation of bone marrow-derived precursors of T lymphocytes. Based largely on its histological appearance, the thymus gland had also been thought to be an endocrine organ. In an attempt to prepare compounds with hormonal properties, aqueous extracts of bovine thymic glands were prepared. A complex mixture of peptides ranging in molecular weight from 1 to 14 kDa was designated thymosin fraction V. Despite the reported capacity of this fraction and some of its component peptides to enhance in vitro or in viuo assays of immune function and enhance immune function in patients with immunodeficiency disease (1, 21, the immunoregulatory function of thymic peptides remains controversial. THYMOSIN
a1
The first peptide in thymosin fraction V to be purified to homogeneity was a highly acidic, 28 amino acid peptide named thymosin o1 (3,4). Many of the biological properties of thymosin fraction V were possessed by this polypeptide. Subsequently, other peptides have also been purified from thymosin fraction V and some of these have also been reported to have biologic activity (5-7). In this review, the origin and biological function of thymosin c+ the best characterized of the peptides in thymosin fraction V, will be considered. As the amino acid sequences of the acidic peptides making up thymosin fraction V were determined, it became clear that a number of peptides shared the same amino terminus, an acetylated serine residue, and had identical amino terminal sequences although they differed in size (8). One peptide was a 24 amino acid polypeptide, thymosin (des 2&28), consisting of the first 24 amino terminal amino acids of thymosin (pi. Another peptide (thymosin ali) was seven amino acids shorter than thymosin al. Larger polypeptides were also isolated which had the 28 amino acids of thymosin 0~~at their amino terminus. This raised the possibility that all of these peptides were derived from a common protein by nonspecific
proteolytic degradation rather than specific cleavage of a precursor protein. This conjecture was supported by the report that intact poly(A) + mRNA isolated from the rat thymus when added to a cell-free protein translation system produced a 16,000-Da product which reacted with an antibody specific for thymosin 0~~(9). The most compelling evidence in support of the origin of many of these peptides from a larger precursor came from the analysis of extracts of fresh rat thymus glands prepared under conditions that inhibited proteolysis. Under such conditions, a 113 amino acid polypeptide reactive with antibodies to thymosin (pi and containing at its amino terminus the 28 amino acid sequence of thymosin oi, but none of the small peptides that shared sequence with thymosin oi, was found (10). Furthermore, purified prothymosin was reported to exhibit many of the biological and immunological activities associated with thymosin 01~including protection against opportunistic infections (11). This purification procedure also yielded a second structurally related, highly acidic polypeptide from rat thymus glands, designated parathymosin (12). This protein inhibited some of the biological activities of prothymosin (11). Thymosin 0~~makes up less than 10% of the total immunoreactive peptide in either serum or red blood cells in uiuo and it has been suggested that this low level is actually the result of partial degradation of prothymosin OLand its leakage from leukocytes (13). In contrast, intact prothymosin cx is extremely abundant in circulating cells (50-70 pmol/ml) and appears to be concentrated in nucleated white blood cells including both polymorphonuclear and mononuclear leukocytes (13). The detection of prothymosin 01 and the failure to detect appreciable levels of thymosin o1 in human blood suggested that thymosin (rl might not actually be a circulating hormone but rather a proteolytic breakdown product of prothymosin (Y. Subsequent studies suggested that prothymosin (Yis not a protein limited to the thymus gland or even a protein limited to cells of immunological origin. Prothymosin (Y was abundant in all tissue examined in195 0090-1229/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
196
SZABO
AND
eluding kidney, liver, and brain (14). Prothymosin (Yis not thymic dependent as it was detected in the tissues of athymic animals as well as in old animals which lack a functional thymus gland. Thus, prothymosin (Yis a protein found in all cell types examined and is not limited to cells of the immune system. THE
STRUCTURE
AND
FUNCTION
OF
WEKSLER
a
b
PROTHYMOSIN
Additional evidence against thymosin oi being a hormone or that prothymosin (Yis a precursor to a hormone comes from sequence analysis of prothymosin (Y cDNA clones (15). There are no additional amino terminal amino acids encoded in the prothymosin (Y mRNA 5’ to the amino terminal acetylated serine that might function as secretory leader sequences. Only the initiator methionine residue is clipped off prior to acetylation of the serine residue (15). While alternative mechanisms for secretion of proteins have been reported (161, such secreted proteins are distinctly unusual. Prothymosin (Y mRNA is also associated with free, cytoplasmic ribosomes, not with rough endoplasmic reticulum-bound ribosomes (17) which would be expected of proteins that are secreted. Prothymosin cxis not only found in a wide variety of tissue types, including thymus, spleen, liver, kidney, brain and muscle, but its mRNA is also detectable in these tissues suggesting that de nouo synthesis of prothymosin (Y occurs in all these cell types (18). These findings suggested that prothymosin (Y may have a more general cellular function and may be required in many different cells and tissues. Proliferating thymocytes express relatively high levels of prothymosin (YmRNA (19). The elevated levels of prothymosin o mRNA seen in early stages of thymocyte development decrease as these cells mature and cease proliferation (19). Mature, circulating T cells do not express high levels of prothymosin (YmRNA under normal conditions (19, 20); however, when T cells are stimulated by mitogen to proliferate, the steady state level of prothymosin (YmRNA increases (20, 21). During the transition from the nondividing GO cells to proliferating cells, the level of prothymosin (Y mRNA increases four- to sixfold and peaks at mid-G1 to the S phase of the cell cycle. This is seen for both T and B lymphocytes (21). As shown in Fig. 1, when T cells, cultured with mitogen, are purified and separated into nonactivated and activated cells, the ratio of prothymosin (YmRNA in activated cells as compared to nonactivated cells is 20 to 1, suggesting that the activity of this protein is expressed at much higher levels in dividing cells. Transformed and proliferating leukemic cells also show increased levels of prothymosin (Y mRNA (19). Prothymosin 01mRNA expression in other cell types including fibroblasts also correlates with their proliferation. Serum-starved, nondividing NIH3T3 cells ex-
< ProT
FIG. 1. Prothymosin u expression in activated T lymphocytes. Shown is a Northern blot of total RNA from PHA-activated and quiescent human T-lymphocytes separated by Percoll gradient centrifugation. A Northern blot of RNA from fractions consisting of activated (a) and quiescent cb) cells was probed with 32P-labeled prothymosin cDNA. The level of prothymosin-specific mRNA in activated cells is >20-fold higher than that seen in the quiescent T cells.
press relatively low levels of prothymosin (Y mRNA. When these cells are induced to proliferate by the addition of serum, the level of prothymosin 01 mRNA increases significantly (22). Similarly, a rapid increase in prothymosin (Yexpression as measured by levels of thymosin immunoreactive peptide is seen in small intestinal crypt cells induced to proliferate by serum stimulation (23). Finally, prothymosin (Yn-RNA expression has also been observed in proliferating hepatocytes in a regenerating rat liver model system (20). As would be expected for a protein required for cell proliferation, prothymosin (Y mRNA expression is reduced in cells which cease dividing. Proliferating murine erythroleukemia cells (MELC) express high levels of prothymosin (YmRNA cultures. These cultures approach saturation density and cease proliferation and the level of prothymosin (YmRNA decreases by nearly two orders of magnitude (P. Szabo, unpublished observations). Similarly, the level of prothymosin 01 mRNA is reduced when HL60 human erythroleukemia leukemia cells are induced to terminally differentiate (24). Thus, the level of prothymosin (Y mRNA reaches negligible levels 1 hr after the exposure of HL60 cells to DMSO, which leads to their differentiation into neutrophils. Exposure of HL60 cells to tetradescanoyl phorbolacetate, TPA, results in a similar loss of prothymosin (YmRNA between 6 and 24 hr after this induction to differentiate into macrophages. The decline in prothymosin (Y mRNA occurs when MELC express their cell-specific differentiation products, e.g., globin. In the case of MELC, which also undergo terminal cell division, there is diminished expression of prothymosin (Y mRNA initially after the addition of the differentiation inducer (e.g., HMl3A), This initial decrease, which is like that seen for HL-60 cells, is followed by a secondary peak of expression at
IS THYMOSIN
a1 A THYMIC
36 to 48 hr after initiation of differentiation with HMBA. The time of the secondary peak varies with cell density and the inducer used. Another protein whose level is modulated by differentiation in these cell types and is associated with proliferation is the c-myc protein. The c-myc gene product, a progression factor, is a nuclear DNA-binding protein which functions as a transacting regulator presumably upregulating genes involved in cellular proliferation (see review (2.5)). Inhibition of the synthesis of c-myc protein prevents the replication of DNA and thereby blocks cellular proliferation. In an effort to determine what genes are specifically regulated by the c-myc protein, an estrogeninducible, recombinant construct containing the c-myc gene was transfected into rat fibroblasts (26). Estrogen induced transfected cells in the stationary phase to enter the cell cycle. Subtractive DNA libraries made from induced cells were screened for sequences that were induced by the expression of the c-myc protein. Initially, only a single clone was obtained which was sequenced and found to be the prothymosin CYgene. Thus, c-myc protein induces prothymosin 01 gene transcription. A potential binding site for c-myc has been detected 1200 base pairs 5’ to the transcriptional start site (P. Szabo, unpublished observations). The expression of c-myc in T cells induced to proliferate occurs early in Gl (27), preceding the appearance of prothymosin (YmRNA in mid to late G1(20,21). The addition of inducers of differentiation to MELC and HL60 cells leads to a fall in c-myc mRNA levels prior to the decrease in prothymosin OLmRNA. Furthermore, in MEL cells, a secondary peak of c-myc expression precedes the secondary peak of prothymosin (Ygene expression. These results are consistent with the direct activation of the prothymosin cx gene by the c-myc gene product. Thus, prothymosin CYappears to play a role in the normal progression of cells through the cell cycle and may, in fact, be required for the replication of DNA. Recently, the addition of antisense oligomers of prothymosin cx mRNA to synchronized human myeloma cells was found to delay cell division (28). The delay, without complete inhibition of cell cycle progression, is due to the instability of the antisense oligomers.
HORMONE?
197
ies are not of high affinity for native prothymosin o, these results may not accurately reflect the distribution of this protein. Direct analysis of the distribution of prothymosin cx in cells was originally done by the injection of labeled prothymosin OLprotein into Xenopus Zueuis oocytes (31). The results of these studies demonstrated that prothymosin CYis rapidly accumulated in the germinal vesicle within 24 hr of injection (31). The related protein, parathymosin, which contains a similar nuclear target peptide, is also seen to accumulate in the germinal vesicle (32). To confirm that prothymosin OLtargets the nucleus, recombinant DNA constructs containing the prothymosin (Y cDNA sequence linked to the human growth hormone (hGH) gene were transfected into HeLa cells (33). The constructs were under the control of the SV40 (large T) promoter and were constitutively expressed in the transfected HeLa cells. The fusion protein derived from this construct could be identified specifically by antibodies to hGH by indirect immunofluorescence. Cells with constructs containing the entire hGH cDNA showed fluorescence in the perinuclear secretory (Golgi) apparatus as expected for a secretory protein (Fig. 2). Cells with the hGH cDNA constructs that lacked the sequences coding for the leader peptide showed diffuse fluorescence throughout the cytoplasm. Cells with the constructs that had the entire prothymosin a or parathymosin sequence flanking the leaderless hGH sequence showed fluorescence concentrated in the nucleus. Thus, both the prothymosin (Y and parathymosin polypeptides have nuclear targeting sequences and the fusion protein is transported to the nucleus. Similar studies using different recombinant constructs confirmed that the prothymosin (Y polypeptide is transported to the nucleus (34). Cells transfected with constructs which lacked the carboxyl terminus, which contained the consensus nuclear targeting sequence, did not transport the protein to the nucleus (34).
The peptide sequence of prothymosin CLis not homologous with any known protein; however, its central, highly acidic stretch of amino acids does show some homology to high mobility group 1 protein (20). This protein is involved in the release of histones from chromatin and it has been proposed that prothymosin (Y PROTHYMOSIN IS A NUCLEAR PROTEIN plays a similar role in the nucleus (20). Since the level 01 mRNA expression in some cells is When the protein sequence of prothymosin (Y was of prothymosin obtained a short stretch of amino acids (KKQKK) near elevated in Gl prior to cell entry into the S phase, it is the carboxyl terminus was found which resembled se- possible that prothymosin cx is required to open chroquences found in proteins that are transported to the matin structure in order to enable DNA to be replinucleus (29). Such a pentapeptide sequence is found in cated. However, the fact that low levels of prothymosin all nuclear targeting proteins examined to date and is CYmRNA are present throughout the cell cycle suggests thought to direct the transport of such proteins to the that this molecule may be also required for other nuclear processes, e.g., an enhancer of RNA transcripnucleus. Direct analysis of the distribution of prothy(Ymay be a secondary transcription mosin (Yin liver cells using antibodies for thymosin (Y~ tion. Prothymosin have identified this protein in both the cytoplasm and factor acting on a limited number of genes needed for the nucleus of cells (30). However, since these antibodproliferation (28).
198
SZABO AND WEKSLER
Isv4oIss
1
hGH
I s
3
Iswl
hGH 13
3
IswI
hGH
1
hPAR4
1
FIG. 2. Nuclear targeting of prothymosin and parathymosin. Recombinant plasmid constructs were prepared to determine the intracellular localization of the thymosins. The reporter gene used in these constructs was human growth hormone cDNA under control of the SV40 large T promoter. The growth hormone product of this clone could be specifically detected using indirect immunofluorescence. The first panel shows this initial construct and a photomicrograph of HeLa cells transfected with this plasmid; fluorescence from the newly synthesized growth hormone is concentrated in the Golgi bodies as expected for a secreted peptide hormone. The second panel shows the results using a second construct in which the secretory signal sequence (ss) has been deleted, as expected, the fluorescence is spread throughout the cytoplasm of the transfected cells. The final panel shows a construct in which parathymosin cDNA has been cloned in proper reading from next to the growth hormone sequence; the fluorescence is found only in the nucleus, suggesting direct nuclear targeting of the parathymosin sequence. The results with constructs bearing prothymosin a, rather than parathymosin cDNA, are identical.
PROTHYMOSIN
GENE
The gene coding for prothymosin o. in humans has been mapped to the long arm of chromosome two (35). It’s structure consists of five exons within about 6.8 kb. The amino acid coding sequences are found in exons l-4; the fifth exon comprises 3’ untranslated sequence. These exons, some of the introns, and the 5’ flanking region have been sequenced (17,35). Numerous, potential &-acting regulatory elements have been identified in the 5’ flanking region (35) of the sequence CACGTG, and these include an E box element located about 1200 base pair 5’ to the start of transcription and known to be recognized by the c-myc:max complex. This later complex has been reported to be the functional transacting c-myc regulatory unit (36). This cisacting regulatory sequence is located about 1.2 kb upstream from the transcriptional start site; binding of the c-myc-max complex at this site may result in direct activation of the prothymosin a gene. In addition to this functional prothymosin a1 gene, there exist at least five prothymosin a-related pseudogenes dispersed in the human genome. These have been partially sequenced and appear to be cDNA pseudogenes (17). The gene coding for prothymosin CYis highly conserved; human cDNA probes crosshybridize to genomic DNA from mice, rats, chickens, fish, and fruit flies, (see Fig. 3). The protein itself has been detected in a wide
variety of species including yeast (37). In the case of murine and rat DNA and to a lesser extent chicken and fish DNA, multiple genomic sites with homology to prothymosin cxare detected by the human probe. It is not clear whether these represent multiple genes in these species or whether these species also contain pseudogenes. In Drosophila, the human prothymosin a cDNA probe appears to detect a unique gene. The evolutionary conservation of sequences for prothymosin a suggests that the function carried out by this protein is of major importance since the nucleotide sequence has been maintained for several hundred million years. CONCLUSION
When first described, the thymosins were thought of as modulators acting as hormones that primarily promoted the proliferation and differentiation of T lymphocytes. It has now become apparent that these compounds, even if they exhibit some immune activities, play a very different biological role. Prothymosin CY,the most well-studied thymosin and the precursor to thymosin ai, has a wide cellular distribution and is not restricted to cells of the immune system. Although its actual role is not known, prothymosin a appears to act primarily in the nucleus of cells and appears to be involved in cellular proliferation. Tran-
IS THYMOSIN
a
b
c
ai A THYMIC
d 5.
23.19.4-
6.
7. 8.
FIG. 3. Evolutionary conservation of the prothymosin Q gene shown is a Southern blot of EcoRI-digested DNA from mouse (lane a), rat (lane b), chicken (lane cl, and Drosophila melunogoster (lane d) annealed with human prothymosin alpha cDNA probe. Hybridization to numerous bands is seen for the two mammalian species presumably due to the existence of multiple pseudogenes. There is less intense hybridization and fewer bands seen for chicken and Drosophila DNA; the relatively dark bands seen in the Drosophila DNA compared to chicken DNA is the result of a fivefold greater DNA in terms of genome equivalents rather than greater homology to the human cDNA probe.
9.
10. 11.
12.
scription of prothymosin (Y may be directly controlled by the c-myc protein, a progression factor whose expression is required for DNA synthesis and cell division. The precise role of prothymosin CYas the cellular proliferation process is currently under intense investigation. ACKNOWLEDGMENTS This work was supported in part by the National Institute on Aging Program Project Grant (AG 00541). We acknowledge the major contributions of Dr. Bernard Horecker, both to our understanding of the thymosins and for his encouragement to enter this field. We thank Drs. L. Graeve, E. Rodriguez-Boulon, M. Clinton, C. Panneerselvam, M. Smith, A. Silverman, M. Mutchnick, and Mr. E. Whittington for information contributing to this review. We are grateful for the secretarial support of Liz Dugan.
13. 14.
15. 16.
17.
REFERENCES Low, T. L. K., and Goldstein, A. L., Thymosins: Isolation, structural studies and biological activities. In “Thymic Hormones and Lymphokines” (A. L. Goldstein, Ed.), Plenum Press, New York, 1984. Goodall, G. J., and Horecker, B. L., “Molecular biology of the Thymosins in Immune Regulation by Characterized Polypeptides,” pp. 283-292, A. R. Liss, New York, 1987. 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., Thymosin alpha-l: Isolation and sequence analysis of an immunologically active thymic polypeptide. Proc. Natl. Acad. Sci. USA 74, 725-729, 1977. Low, T. L. K., Thurman, G. B., Chincarini, C., McClure, J. E., Marshall, G. D., Hu, S-K., and Goldstein, A. L., “Current status
18.
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20.
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HORMONE?
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of thymosin research: Evidence for the existence of a family of thymic factors that control T-cell maturation”. Ann N.Y. Acud. Sci. 332, 33-48, 1979. Schulof, R. S., and Goldstein, A. L., Thymosins and other thymic hormones. In “The Lymphokines: Biochemistry and Biological Activity” (J. W. Hadden and W. E. Stewart, Eds.), p. 379, Humana Press, Clifton, NJ, 1981. Low, T. L. K., Hu, S-K., and Goldstein, A. L., Complete amino acid sequence of bovine thymosin B4: a thymic hormone that induces terminal deoxynucleotidyl transferase activity in thymocyte populations. Proc. Nutl. Acad. Sci. USA 78, 1162-1166, 1981. Erikson-Viitaner, S., Ruggieri, S., Natalini, P., and Horecker, B. L., Thymosin B,,, a new analog of thymosin B, in mammalian tissues. Arch B&hem. Biophys. 225, 407-413, 1983. Cardarella, J., Goodall, G. J., Felix, A. M., Heimer, E. P., Salvin, S. B., and Horecker, B. L., Thymosin alpha 2, a peptide related to Thymosin alpha 1 isolated from calf thymosin fraction 5. Proc. Natl. Acad. Sci. USA 80, 7424, 1983. Freire, M., Hannappel, E., Rey, M., Freire, J. M., Kido, H., and Horecker, B. L., Purification of thymus mRNA coding for a 16,000-dalton polypeptide containing the thymosin alpha-l sequence. Proc. Natl. Acad. Sci. USA 78, 192-195, 1981. Haritos, A. A., Blacker, R., Stein, S., Cardarella, J., and Horecker, B. L., Primary structure of rat thymus prothymosin alpha. Proc Natl. Acad. Sci. USA 82, 343-346, 1985. Pan, L-X., Haritos, A. A., Wideman, J., Komiyama, T., Chang, M., Stein, S., Salvin, S. B., and Horecker, B. L., Human prothymosin alpha: Amino acid sequencing and immunologic properties. Arch. Biochem. Biophys. 250, 197-201, 1986. Haritos, A. A., Salvin, S. B., Blacker, R., Stern, S., and Horecker, B. L., Parathymosin alpha: A peptide from rat tissues with structural homology to prothymosin. Proc. Natl. Acud. Sci. USA 82, 1050-1053, 1985. Panneerselvan, C., Haritos, A. A., Cardarella, J., and Horecker, B. L., Prothymosin alpha in human blood. Proc. Natl. Acud. Sci. USA 84, 4465-4469, 1987. Haritos, A. A., Tsolas, O., and Horecker, B. L., Distribution of prothymosin alpha in rat tissues. Proc. Natl. Sci. USA 81, 13911393, 1984. Goodall, G. J., Dominquez, F., and Horecker, B. L., Molecular cloning of cDNA for human prothymosin alpha. Proc. N&Z. Acad. Sci. USA 83, 8926-8928, 1986. Matsushima, K., Taguchi, M., Kovacs, E. J., Young, H. A., and Oppenheim, J. J., Intracellular localization of human monocyte associated interleukin 1 (IL 1) activity and release of biologically active IL 1 from monocytes by trypsin and plasmin. J. Immunol. 136, 2883-2891, 1986. Eschenfeldt, W. H., Monrow, R. E., and Bergen, S. L., Isolation and partial sequencing of the human prothymosin alpha gene family: Evidence against export of the gene products. J. Biol. Chem. 264, 75467555,1989. Clinton, M., Frangou-Lazaridis, M., Panneerselvan, C., and Horecker, B. L., Prothymosin alpha and parathymosin: mRNA and polypeptide levels in rodent tissues. Arch. ofBiochem. Biophys. 269, 256-263, 1989. Gomez-Marquez, J., Segade, F., Dosil, M., Pichel, J. S., Bustelo, X. R., and Freire, M., The expression of prothymosin alpha gene in T lymphocytes and leukemic lymphoid cells is tied to lymphocyte proliferation. J. Biol. Chem. 264, 8451-8454, 1989. Bustelo, X. R., Otero, A., Gomez-Marquez, J., and Freire, M., Expression of the rat prothymosin alpha gene during T-lymphocyte proliferation and liver regeneration. J. Biol. Chem. 266, 1443-1447,199l. Szabo, P., Ehleiter, D., Whittington, E., and Weksler, M., Pro-
200
SZABO thymosin alpha T lymhocytes. 1992.
expresion
occurs
during
Gl in proliferating
AND B or
Biochem. Biophys. Res. Comm., 185, 953-959,
22. Eschenfeldt, W. H., and Berger, S. L., The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: Evidence using a cloned cDNA. Proc. Natl. Acad. Sci. USA 83, 9403-9407, 1986. 23. Conteas, C. N., Mutchnick, M. G., Palmer, K. C., Weller, F. E., Luk, G. D., Naylor, P. H., Erdes, M. R., Goldstein, A. L., Panneerselvan, C., and Horecker, B. L., Cellular levels of thymosin immunoreactivepeptides are linked to proliferative events: Evidence for a nuclear site of action. Proc. Natl. Acad. Sci. USA 87, 3269-3273, 1990. 24. Smith, M. R., Silverman, A., Szabo, P., Kohler, W., Nath, R., and Mutchnick, M. G. Prothymosin alpha gene expression is downregulated in differentiating HL-60. Submitted for publication. 25. Luscher, B., and Eisenman, R. N., New light on Myc and Myb: Part 1. Myc. Genes Dev. 4, 2025-2035, 1991. 26. Eilers, M., Schiron, tivates transcription 133-144,1991.
S., and Bishop, J. M., of the a-prothymosin
The MYC protein acgene. EMBO J. 10,
27. Gamble, D., Schwab, R., Weksler, M.E., and Szabo, P., The ageassociated in vitro proliferative defect(s) in human T-lymphocytes precedes c-myc gene activation. J. Immunol. 144, 35693573,199o. 28. Sburlati, A. R., Manrow, R. E., and Berger, S. L. “Prothymosin alpha antisense oligomers inhibit myeloma cell division”. Proc. Natl. Acad. Sci. USA 88, 253-257, 1991. 29. Gomez-Marquez, J., and Segade, F., Prothymosin alpha is a nuclear protein. FEBS Lett. 226, 217-219, 1988. Received
June
25, 1992;
accepted
July
8, 1992
WEKSLER Komiyama, T., Pan, L-X., Haritos, A. A., Wideman, J. W., Pan, Y-CE., Chang, M., Rogers, I., and Horecker, B. L., The primary structure of rat parathymosin. Proc. Natl. Acad. Sci. USA 83. 1242-1245,1986. 30. Conteas, C., Su, Y-L., 3. A1151, 1989
Palmer,
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M.,
FASEB J
31. Watts, J. D., Cary, P. D., and Crane-Robinson, C., Prothymosin alpha is a nuclear protein. FEBS Lett. 245, 17-20, 1989. 32. Watts, J. D., Cary, P. D., Sautiere, P., and Crane-Robinson, C Thymosins: Both nuclear an cytoplasmic proteins. Eur. J Biothem. 192, 643-651, 1991. 33. Clinton, M., Graeve, L., El-Dorry, H., Rodriguez-Bouion, E.. and Horecker, B. L., Evidence for nuclear targeting of prothymosin and parathymosin synthesized in situ. Proc. Natl. Acad. Sci.
USA 88, 6608, 1991. 34. Manrow, R. E., Sburlati, A. R., Hanover, J. A., and Berger, S. L., Nuclear targeting of prothymosin alpha. J. Biol. Chem. 206. 3916. 1991. 35. Szabo, P., Panneerselvam, C.. Clinton, M., Frangou-Lazaridis. M., Weksler, D., Whittington, E., Macera, M. J., Grzeschik. K-H., Selvaknmar, A., and Horecker, B. L., Localization of the prothymosin alpha gene to chromosome 2 in humans and organization of its promoter region. Submitted for publication. 36. Blackwood, G. C., and Eisenman, R. N., Max: A helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with myc. Science 251, 1211-1217, 1991. 37. Makarova, YR, Grebenshikov, N., Egorov, C., Vartapetian, A,. and Bogdonov, A., Prothymosin alpha is an evolutionary conserved protein covalently linked to a small RNA. FEBS Lett. 257. 247-250. 1989.