Thymosin α1 does not promote growth or oncogenic transformation

Int. J. Immunopharmac., Vol. 18, No. 5, pp. 321-327, 1996 Copyright © 1996InternationalSocietyfor Immunopharmacology Publishedby ElsevierScienceLtd. Printedin Great Britain 0192~)561/96$15.00+ .00

Pergamon

PII: S0192-0561(96)00032-X

T H Y M O S I N ~1 DOES N O T P R O M O T E G R O W T H OR O N C O G E N I C TRANSFORMATION PAUL H. NAYLOR*t, MITCHELL R. SMITH*, MILTON G. MUTCHNICK*, CARTHA W. NAYLOR*, JULIE DOSESCU*, MAGDALENA SKUNCA* and JEFFREY A. MOSHIER:~ *Division of Gastroenterology, Department of Internal Medicine, Wayne State University School of Medicine, Detroit, MI 48201, U.S.A.; and :~Centerfor Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, U.S.A. (Received 29 November 1995)

A~tract--Thymosin al(Tctl) is an immune modulatory peptide which has been evaluated in a variety of clinical trials. Although no in vivo adverse effects, including enhancement of tumor growth, have been noted, in vitro studies suggestinga role for Ttzl in cell growth have been reported. The studies presented in this report evaluated both exogenouslyadded Tot1 and endogenouslyexpressed T:t 1 as factors which could either promote growth of tumor cells or induce transformation. No effect of exogenous Tatl on cell growth was found. NIH3T3 cells transfected with cDNA for the precursor ProThymosin ~t (ProT00 expressed elevated levels of authentic Tctl but did not demonstrate either enhanced proliferation in liquid culture or transformation as defined by the loss of contact inhibition or anchorage independent growth in soft agar. Thus these studies argue against the hypothesis that Tal is either an intracellular or extracellular growth promoter. Copyright © 1996 International Society for Immunopharmacology Keywords: thymosin ctl, thymic peptides, Prothymosin.

Thymosin 0tl (T~tl) is a peptide originally isolated from the thymus on the basis of its activity in the induction of mature T cell markers on precursor lymphocytes (Goldstein et al., 1977). Subsequent studies demonstrated a more extensive range of activities leading to its designation as a biological response modifier and extensive testing in vitro, in vivo and in clinical trials (Schulof et al., 1987; Schulof et al., 1985; Gravenstein et al., 1989; Schen, 1987; Mutchnick et al., 1991; Andreone et al., 1993). Most clinical trials utilize Tctl as an immune enhancing agent, either alone or in combination with other agents. Successful trial results have been published using Tctl as: (a) a follow-up to irradiation to prevent relapse in lung cancer patients (Schulof et al., 1985); (b) an enhancer of titers to the flu vaccine in the elderly (Gravenstein et al., 1989); (c) an enhancer of the hepatitis B vaccine in dialysis patients (Schen, 1987); and (d) an enhancer of the clearance of chronic HBV infection (Mutchnick et al., 1991; Andreone et aL, 1993). Although no significant adverse effects have been reported for T~tl and no enhancement of tumor growth has been observed in the cancer trials, studies

evaluating the role of T~tl in cell growth have yielded mixed results (Eishenfeldt & Berger, 1986; Bustelo et al., 1991; Szabo et al., 1992; Smith et al., 1993; Conteas et al., 1990; Sburlati et al., 1993; Zalvido et al., 1992; Sburlati et al., 1991; Gomez-Marquez & Segade, 1988; Watts et al., 1989; Clinton et al., 1991; Manrow et al., 1991; Appel et al., 1993; Oates et al., 1988). The messenger RNA coding for the putative precursor Prothymosin ct(ProTct), as well as the ProTct peptide, Tctl and additional uncharacterized immunoreactive thymosin ctl like antigens (TLA) have all been identified in a variety of tissues, cells in culture, supernatants from cultured cells and serum (Eishenfeldt & Berger, 1986; Bustelo et al., 1991; Szabo et al., 1992; Smith et al., 1993; Conteas et al., 1990). The principal support for a role of Tatl in cell growth comes from reports that Tatl, ProT~ and mRNA for ProTct/Tctl increase with cell growth induced by the addition of serum to serum starved cells (Eishenfeldt & Berger, 1986; Szabo et al., 1992; Conteas et al., 1990). Additional evidence is derived from observations that the precursor protein, ProTa (and perhaps Tot1) translocate to the nucleus (Gomez-Marquez & Segade,

tAuthor to whom correspondence should be addressed at current address: Detroit VA Medical Center--151, 4646 John R, Detroit, MI 48201, U.S.A. 321

322

P.H. NAYLOR et aL

1988; Watts et aL, 1989; Clinton et al., 1991; Manrow et al., 1991), and that ProTa antisense but not sense constructs decreased cell proliferation (Sburlati et al., 1991). In contrast, other studies reported that both the message for ProTct and the levels of ProTct and its phosphorylation were stable throughout the cell cycle (Sburlati et al., 1993; Zalvido et al., 1992). These conflicting reports of a role for ProTct/Tatl in the initiation of the growth of tumor cells, as well as the increased use of Tctl in clinical trials, led us to examine whether the biologically active peptide used in clinical trials (Tatl) increased cell proliferation. These studies actively evaluated the cell growth response by adding exogenous Tctl and intracellularly elevating Ted via gene transfection. In the first set of studies T a l was added to cells in culture to determine whether it would cause an increase in cell growth. The demonstration that Ted does not cause the growth of tumor cells would be consistent with the role of Tctl as an exogenous inducer of cell maturation/activation rather than as a growth inducing cytokine. In the second set of studies, the cDNA coding for ProT~ was transfected into NIH/3T3 cells to determine whether intracellular T~l/ProTct had oncogenic transformation potential and or cell growth potentiating activity. The NIH/3T3 cells were selected because they are the classic cell line for demonstrating effects of agents on transformation and because previous studies utilized them as a model to demonstrate changes in m R N A for ProTa following serum refeeding (Eishenfeldt & Berger, 1986).

EXPERIMENTAL PROCEDURES

Cell growth curves Cells were grown in media supplemented with serum appropriate for the cell type. Cell culture reagents were from Gibco Inc. (Grand Island, NY, U.S.A.) with other reagents from Sigma Inc. (St. Louis, MO, U.S.A.) unless stated otherwise. T:tl was the gift of Alpha One Biomedicals (Foster City, CA, U.S.A.). Cells evaluated were HL-60, CEM, DLD1, MCF-7, HTC-8, and NIH3T3. All were from the ATTC (Bethesda, MD, U.S.A.) and a complete description of the cells and recommended culture conditions is available from their catalogue and accompanying references. For the experiments cells were plated at densities appropriate for rapid initiation of log phase growth. Cell proliferation was assessed by visual counting of viable cells using a hemocytometer and trypan blue, or by the addition of 3H-thymidine (2 uCi/ml) 12 h before harvest. For

3H-thymidine analysis, cells were collected on filter paper using an automated cell harvester and radioactivity determined after the addition of scintillation cocktail. Varying concentrations of fetal calf serum were used to determine whether the addition of Tetl could substitute for growth factors in the serum. Transfection o f NIH/3T3 cells The ProTct cDNA coding region was generated by RNA-PCR of HL-60 cell m R N A using PCR primers with recognition sites for cloning downstream of a Rouse Sarcoma Virus promoter. Sequencing analysis of the PCR product demonstrated no deviations from the published sequence for fibroblast ProTct (Szabo et aL, 1992). The ProTet cDNA contained the full length coding region with no 5' or 3' untranslated sequences. Restriction digestion of the PCR product and PCR/RSV plasmid permitted ligation of the ProTct cDNA behind the RSV promoter in the same vector as the neo gene. NIH/3T3 cells were transfected with the two constructs using calcium phosphate precipitation and the presence of the inserts was verified by growing the cells in geneticin. The message produced by the insert is predicted to be shorter than the native message, yielding two m R N A hybridizing bands in successfully transfected cells (one for native and one for the insert) on Northern blots. The full length c D N A 32p labeled probe was used in the Northern blots. The NIH/3T3 cells with the RSVneo construct were designated NRV while the cells with the ProTet insert are designated NPTet. Anchorage independent and cell foci growth assays Transformation due to transfection with the constructs was assessed as previously described using either soft agar growth or foci formation (Moshier et al., 1993). Cells were plated at 1000 cells per plate into soft agar or media and incubated for 10-15 days. Media was added every 3 days. The number of colonies exceeding 50 cells per colony or the number of foci was counted. Extraction o f T L M The extraction of T~l/ProTct and other Ta like antigens (TLA) was as previously described (Naylor et al., 1992; Naylor et aL, 1994). In brief cell extracts were prepared by lysing the cells in water using three 10 s bursts of a sonicator set at 80%. After centrifugation at 10,000 g to remove debris, the extract was passed through a Waters C18 Sep-Pak. The TLA was eluted by the addition of 25% acetonitrile, a concentration previously shown to elute authentic Tetl and ProTct from spiked tissue and serum samples as

Cell Growth and Thymosin ~1 well as thymus and peripheral blood lymphocyte extracts (Naylor et al., 1992). ELISA assays were used to confirm the presence of TLA's in the eluate. Recoveries as assessed by the HPLC using T~tl standard were 75-85%. The acetonitrile in the samples was removed by vacuum and the sample reconstituted using HPLC grade water.

3

\,

A

-

ELISA

The ELISA for Ted was performed as previously reported (Naylor et al., 1994; Weller et al., 1988). The ELISA utilized a rabbit antibody specific for T0tl. Samples were incubated with the antibody overnight at 4°C and transferred to Tctl coated microtiter plates for a 40 rain incubation at 37°C. The assay thus measures the antibody which has not bound to Ted in the sample in the overnight incubation. The presence of antibody was determined by sequential addition (with washes in between) of biotinylated goat anti-rabbit, avidin-biotinylatedalkaline phosphatase and p-nitrophenyl phosphate substrate. Synthetic Tctl was used as the standard. ProTct purchased from Peninsula Labs (Foster City, CA, U.S.A.) was not detected in the assay even at concentrations as high as 1000 ng/ml (vs minimal detection of Tal at 100 pg/ml).

RESULTS Typical results from studies evaluating in vitro effects on tumor cell growth are reported in Fig. 1. The three tumor cell lines that were grown in the presence of varying concentrations of Ta 1 were breast (MCF-7), colon (DLD- 1) and ileocecal (HCT-8) adenocarcinomas. No dose responsive effect on cell growth was detected at T a l concentrations between 1 Ixg and 0.1 pg/ml. Since fetal calf serum supplemented media contains thymosin al-like antigens (TLA) (Naylor, unpublished observation), cells were also tested for growth in lower concentrations of FCS (i.e. Tcz1 levels less than 100 pg/ml). In the low serum experiments cells were grown in 0.5% serum with T~I con-

~

i

] ~i/r\\

Sizing H P L C

The reconstituted sample (either from the Sep-Pak or after initial purification on a reverse phase column) was injected on to a Beckman TSK 2000 sizing column equilibrated with a 0.01 M sodium acetate, 0.15 M sodium chloride, pH 6.0 buffer which was also used to elute the sample. The eluted material was collected in 0.3 ml aliquots and assayed directly by the ELISA. Known molecular weight standards were used to calibrate the column (Naylor et al., 1992).

323

o 0.1

. ~--, ~ ...................... i

10

DLD-1

\\\

]

VX .-'" I ~. . < " / , ' " i -t~'~-'-'"'~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

1000

Thymosin o l ~ -

"~

iI

-e--

10000

100000

1000000

li*OTf

( polml )

MCF-7

--8-

HTC

Fig. I. Thymosin ctl effect on cell growth using three cells lines previously reported to be growth responsive.Thymosin ~tl (Tctl) was added at the doses indicated to cells and cell growth determined at day 3 by tritiated thymidine uptake. The results are expressed as stimulation index (value with Tot1/value for media control). The error bars represent standard error for the triplicate cultures and are included only at representative points to allow for clarity of the graph. The box defines the growth which would be required for a significant (2 fold) increase or decrease from the media alone cultures. centrations from 100 to 0.1 ng/ml with no difference from controls cells on the basis of trypan blue dye exclusion counting of viable cells (not shown). Cells tested in this manner include IEC-6 intestinal crypt cells, 3T3 mouse fibroblast cells, CEM lymphoma cells, and HepG2 hepatoblastoma cells. In other studies, media was supplemented with selenium, insulin and transferrin rather than serum and growth assessed by 3H-thymidine. The cells evaluated were human lymphomas of T cell (CEM) and B cell (DB) origin. Tctl concentrations from 100 pg/ml to 1 pg/ml were used with no effect on cell growth (data not shown). Although exogenous Tctl is not a growth factor as demonstrated by the studies using cells which in most cases were already transformed, the effect of increasing intracellular Ta 1 (either by uptake or intracellular synthesis) required assessment. To accomplish this second goal, cells were engineered to contain elevated intracellular ProT~ mRNA by transfecting NIH/3T3 cells with ProTct cDNA coding sequences driven off Rouse Sarcoma virus promoter. NIH/3T3 cells were used because of their propensity to transform when transfected with oncogenes and the fact that increased mRNA for Ted has been described following serum starvation and refeeding. Designated NPTct, the ProTct transfected cells expressed two bands on Northern blots when probed with the eDNA for ProTQt (Fig. 2). The lower molecular weight band is consistent with the size of the inserted message since no upstream regulatory regions are present in the

P. H. NAYLOR et al.

324

200

Identification of ProThymosin a mRNA in Transfected Cells ~

/

lOO

=

5

Native Transfected

z

F

z

z

Fig. 2. Northern blot analysis of cells using P32 labeled ProTa cDNA. The control cells contain a viral promoter and resistance gene alone (RNV) while the overexpressing cells contain an additional segment upstream representing the ProTa cDNA (NPT~). The two lanes represent cells incubated with or without thymosin al (0.1 p.g/ml) and harvested at log phase. Equal amounts of nucleic acid were loaded as confirmed by hybridizing with 18S and ethidium bromide staining. *Indicates cells grown in presence of Tal ( 10 ~g/ml).

truncated c D N A construct. Consistent with previous reports, the e n d o g e n o u s m R N A for P r o T ~ was a b u n d a n t a n d easily visualized in b o t h cells. The m R N A representing the transcription of the inserted P r o T ~ c D N A was present in lower a m o u n t s a n d neither was modified by the addition of T~I (0.1 ~tg/ml) to the cells in culture. Cells in log phase were harvested, a n d the T L A in the cell extracts were measured by ELISA. The P r o T ~ transfected cells (designated N P T ~ ) h a d only slightly more T L A then the control cells (designated N R V ) (178.3+ 22.7 vs 99.5+_ 10.5 T~I ng equivalents/ml in a typical experiment). The P r o T ~ transfected cells were c o m p a r e d to the control in a tissue culture growth assay. There was no growth a d v a n t a g e for the N P T ~ cells (Fig. 3). Cells were also evaluated for two parameters o f unregulated cell g r o w t h - - s o f t agar growth a n d foci formation. The control cells ( N R V ) a n d the transfected cells (NPT~) b o t h grew poorly in soft agar. The low growth was similar to that o f the parental 3T3 (data not shown). In the alternative experiment to evaluate t r a n s f o r m a t i o n , cells were plated at low density and f o r m a t i o n o f foci determined (Fig. 4). The two sets o f cells transfected with the expression vectors ( N L K a n d R N V ) grew as monolayers, as did the N P T ~ cells. This was in con-

6

7

8

9

Time Post Seeding ( d a y s ) +

NRV

/~

NPTa

Fig. 3. Growth of transfected cells on plastic. NIH/3T3 cells transfected with the expression vector and antibiotic resistance gene (NRV) or the vectors with an upstream ProTe~ cDNA (NPT~) were plated at 5000 cells per well in a 6 well plate and the media changed on days 4 and 7. Cell counts were at the times indicated. The results are for triplicate cultures.

NODC

NLK

NPToL

NRV

Fig. 4. Colony formation as a criterion for transformation was assessed in plastic by plating cells at low density and staining for colonies. Control NIH 3T3 cells containing expression and resistance genes are designated as NRV and NLK. These control cells do not form colonies either on plastic (shown) or in soft agar (not shown) The cells transfected with the ProT~ insert are designated NPT~ and also show no colony formation on plastic (shown) or in soft agar (not shown). For comparison, cells transfected with the ornithine decarboxylase gene (designated NODC) formed significant numbers of colonies on plastic (shown) and in soft agar (not shown).

Cell Growth and Thymosin ctl trast to the NODC cells which were transfected with the gene for ornithine decarboxylase and formed readily identifiable foci (Moshier et al., 1993). Since the presence of extra copies of m R N A and a slight increase in cross-reactive antigen (TLA) does not necessarily mean an increase in authentic Tatl in the cells the TLA was characterized using an H P L C ELISA method. The cell extracts were subjected to sizing HPLC and the TLA in the fractions measured by ELISA (Fig. 5). Multiple ELISA reactive peaks representing minimally detectable levels of cross-reactive material were detected in the various fractions obtained from the HPLC sizing column when control NRV cell extracts were analyzed. The NPT~ cells which had the additional ProTat cDNA had an increase (approximately 4 fold) in the amount of authentic T~tl as defined by a retention time of the immunoreactive material identical to the synthetic T~I standard. Cell extracts were adjusted to reflect differences in cell numbers prior to injection into the HPLC for analysis.

DISCUSSION Addition of Tctl to cells in culture does not induce cell proliferation. Thus Tctl is not a growth factor. sl

Tal

7 6

4 Z

~ 3 2

1

0 12

.I~

.a

.g

la.2

.a

.|

14.1

.4

.7

~e

.s

.e

.~

~¢i

.|

Retention Time --o~

I~l'o

-~-

NHV

Fig. 5. Sizing HPLC analysis of intracellular extracts from over expressing (NPTct) and control (NRV) cells. Cell extracts were subjected to sizing HPLC, fractions collected at the times indicated and the presence of the TLA determined by ELISA. The NPTQtcells had a 4 fold increase in of Tctl as defined by the presence of the material at the appropriate molecular weight. Additional immunoreactive peaks with lower molecular weights were present in both cells. The data are representative of five HPLC-ELISA studies and are presented as the results of three ELISA assays of one HPLC run. As previously reported the retention time for Ted as defined by molecular weight standards was 9000 daltons, significantly higher than the calculated weight of 3100 but still smaller than ProTct which also has a higher molecular weight as defined by the sizing column (Naylor et al., 1992).

325

Addition of Tctl to NIH/3T3 cells or the transfection and continuous expression of ProTat m R N A in NIH/3T3 cells does not result in either an increase in growth or transformation as defined by growth in soft agar or foci formation. These results are in contrast to two reports of an increase in growth following T0tl addition (Appel et al., 1993; Oates et al., 1988). In one study using MCF-7 breast cancer cells the Tctl increase in proliferation was small (92%) and statistically most significant only when the cell growth was stimulated by 17-beta estradiol(Oates et al., 1988). Thus, as the authors suggested, although Ted may influence the effect of other growth factors (such as estrogen in estrogen dependent cells), it may not be a growth factor per se. In the other paper reporting the full details of a preliminary abstract, our data that HTC cells did not respond to Tat with increased cell growth was confirmed. Their studies differed with those presented here in that they reported an increase in growth of the DLD-1 cells. The increase they report in DLD-1 cell numbers (1.3-2.4 fold increase at doses of 1 ug/ml and 0.1 ng/ml; p < .05) were restricted to a narrow phase of the growth cycle (i.e. only on days 7 and 10 post plating). Although there is no apparent reason for the differences, increases in those studies were small (2-4 fold) but statistically significant (Appel et al., 1993). The studies presented in our paper used 3H-thymidine rather than manual cell counts providing for a more accurate and perhaps less subjective analysis. With respect to the biology ofTatl, one controversy relevant to these studies is whether Tatl or the precursor ProT~t is the biologically important peptide (Eishenfeldt & Berger, 1986; Bustelo et al., 1991; Szabo et al., 1992; Smith et al., 1993; Conteas et al., 1990; Sburlati et al., 1993; Zalvido et al., 1992; Sburlati et al., 1991; Gomez-Marquez & Segade, 1988; Watts et al., 1989; Clinton et al., 1991; Manrow et al., 1991; Freire et al., 1985; Franco et al., 1992; Clinton et al., 1992; Haritos et al., 1985; Eishenfeldt et al., 1989; Tsitsiloni et al., 1993). In the procedure used for the original isolation of Tctl from calf thymus, the tissue was initially homogenized in phosphate buffer and the Tctl was the major peptide isolated on the basis of the bioassay (Goldstein et al., 1977). In vivo, the 28 amino acid Tatl must be produced from a presumed precursor Prothymosin 0t (ProTat) (Eishenfeldt & Berger, 1986; Eishenfeldt et al., 1989). Although not directly demonstrated, this hypothesis is based on the genomic and e D N A studies demonstrating only m R N A of a size consistent with the precursor is present in ceils. The molecular biology is also confirmed by studies reporting the presence of ProT~t and T a l in extracts from large amounts of

326

P.H. NAYLOR et al.

tissue where rapid boiling of the tissues is used to prevent degradation of the precursor (Freire et aL, 1985; Franco et al., 1992; Clinton et al., 1992; Haritos et al., 1985; Eishenfeldt et al., 1989). Additional confusion is a result of the reports that both ProT~t and Tctl have similar biological activities(Freire et al., 1985; Franco et al., 1992; Clinton et al., 1992; Haritos et al., 1985; Eishenfeldt et al., 1989). Tctl has been used in more clinical studies since it is the bioactive fragment and can be easily synthesized in large amounts. Although ProTct is predicted by the nucleotide sequence of the c D N A and the amounts o f m R N A are abundant it has been difficult to isolate large amounts from tissues and is not readily available for study (Freire et al., 1985; Franco et al., 1992; Clinton et al., 1992; Haritos et al., 1985). In the studies presented here, the E L I S A utilized is specific for TQtl and does not recognize ProTct (abstract # 254 F A S A B Proceedings, 1995). This is presumably because the Tctl epitopes of ProTct are not accessible to the antibody in solution. Thus cross-reactive peptides with unknown retention times or retention times different from Tctl are designated T L A s since they cross-react in the E L I S A and can not be ProTa. If ProTct was present in extracts and subsequently in the H P L C fractions, it would not be detected. Thus, the lack of ProT~ in the H P L C fractions from the two cell lines does not indicate a lack of ProTct in the cell extracts, just the inability of the assay to measure ProTat. In the transfected cells used in the studies presented here, the m R N A produced from the insert was increased to levels similar to several other transfection

experiments with other genes although the levels were still lower than the endogenous message (Moshier et al., 1993). The NPTct cells produced authentic Tctl at levels approaching 4 fold higher than the N R V control cells. These studies do not address the issue of ProTct as a precursor to TQtl or even as a significant component of the cell extracts since the E L I S A assay does not measure ProT~t at the levels predicted in the cells and ProTct was not available in large enough amounts for the exogenous addition experiments. It is assumed however that the presence of elevated levels of the authentic Tctl in the cells with the inserted gene, confirms the presence in the cells of a pathway which will process the 113 amino acid precursor to the 28 aa peptide. These studies demonstrate that Tctl not an exogenous growth factor, and it is also not an endogenous growth promoter. Whether changes in m R N A ' s for ProT~t which correlate with cell growth means a role for the larger ProTat molecule or other forms of T L A in cell growth remains unclear. The final determination of this issue probably awaits the utilization in similar studies comparing m R N A and protein levels using an assay which is specific for the P r o T a protein and in which the T~tl does not cross-react (Tsitsiloni et al., 1993).Clinton et al., 1989 Acknowledgements--The authors wish to thank Doug Sonneck, Luther Burse, and Sheila Kilahani for their technical assistance. They thank Allan L. Goldstein for his support and critical comments on the manuscript. Financial support for these studies came as gifts and grants from Alpha One Biomedicals, Bethesda, MD and SciClone Pharmaceuticals Inc., San Mateo, CA.

REFERENCES

Andreone, P., Cursaro, D., Gramenzi, A., Miniero, R., Manzin, A., Severini, R., Franzone, J. S., Clementi, M., Sprovieri, G. & Gasbabrini, G. (1993) Preliminary results of Thymosin ctl vs IFN-ct treatment ill patients with HBeAg negative and HBV-positive chronic active hepatitis. Int. J. Immunotherapy, 126, 201-206. Appel, J. L., Arnold, E. B., Conteas, C., Bergsman, K. L. & Luk, G. (1993) Relationship of thymosin ctl immunoreactivity and exogenous stimulation on proliferation of Human colon cancer cell lines. Biochem. Arch., 9, 355-363. Bustelo, X. R., Otero, A., Gomex-Marquex, J. & Freire, M. (1991) Expression of the rat prothymosin at gene during T lymphocyte proliferation and liver regeneration. J. Biol. Chem., 266, 1443. Clinton, M., Frangou-Lazaridis, M., Panneerselvam, C. & Horecker, B. L. (1989) Prothymosin at and parathymosin: mRNA and polypeptide levels in rodent tissues. Arch. Biochem Biophys., 269, 256-263. Clinton, M., Graeve, L., EI-Dorry, H., Rodriquez-Boulan, E. & Horecker, B. L. (1991) Evidence for nuclear targeting of prothymosin ct and parathymosin synthesized in situ. Proc. Natl. Acad. Sci. USA, 88, 6608~614. Conteas, C. N., Mutchnick, M. G., Palmer, K. C., Weller, F. E., Luk, G. D., Naylor, P. H., Erdos, M. R., Goldstein, A. L., Panneerselvam, C. & Horecker, B. L. (1990) Cellular levels of thymosin immunoreactive peptides are linked to proliferative events: Evidence for a nuclear site of action. Proc. Natl. Acad. Sci. USA, 87, 3269-3273. Eishenfeldt, W. H. & Berger, S. L. (1986) The human prothymosin gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA. Proc. Natl Acad. Sci., 83, 9403-9407. Eishenfeldt, W. H., Manrow, R. E., Krug, M. S. & Berger, S. L. (1989) Isolation and partial sequencing of the human prothymosin ctl gene family. J. Biol. Chem, 264, 7546-7555. Franco, F. J., Diaz, C., Barcia, M. & Freire, M. (1992) Thymosin 51 is a native peptide in several tissues. Biochem. et Biophysica Acta, 1120, 43-48.

Cell Growth and Thymosin atl

327

Freire, M., Rey-Mendez, M., Gomez-Marquez, J. & Arias, P. (1985) Evidence for the synthesis of thymosin ~1 by thymocytes and for the production of this peptide by natural processing. Arch. Biochem. Biophys., 239, 481)--487. Goldstein, A. L., Low, T. L. K., McAdoo, M., McClure, J., Thurman, G. B., Rossio, J. L., Lai, C-Y., Chang, D., Wang, SS., Harvey, C., Ramel, A. H. & Meinhofer, J. (1977) Thymosin at1: isolation and sequence analysis of an immunologically active thymosin polypeptide. Proc. Natl. Acad. Sci. USA, 74, 725-729. Gomez-Marquez, J. & Segade, F. (1988) Prothymosin alpha is a nuclear protein. FEBS Lett., 226, 217-219. Gravenstein, S., Duthie, E. H., Miller, B. A., Roecker, E., Dainka, P., Prathipati, K. & Ershler, W. B. (1989) Augmentation of influenza antibody response in elderly men by thymsoin alpha 1. J. Am. Geront. Soc., 37, 1-8. Haritos, A. A., Blacker, R., Stein, S., Caldarella, J. & Horecker, B. L. (1985) Primary structure of rat thymus prothymosin ct. Proc. Natl. Acad. Sci. USA, 82, 343-346. danrow, R. E., Sburlati, A. R., Hanover, J. A. & Berger, S. L. (1991) Nuclear targeting of prothymosin ~. J. BioL Chem., 266, 1443-1451. Moshier, J. A., Dosescu, J., Skunca, M. & Luk, G. D. (1993) Transformation of NIH/3T3 cells by ornithine decarboxylase overexpression. Cancer Res., 53, 2618-2622. Mutchnick, M. G., Appleman, H. D., Chung, H. T., Argone, E., Gupta, T. P., Cumming, C. G., Waggoner, J. G., Hoffnagle, J. H. & Shafritz, D. A. (1991) Thymosin treatment of chronic hepatitis B: a placebo-controlled pilot study. Hepatoloyy, 14, 409-415. Naylor, P. H., Oates, K. K., Coss, M. C., Erdos, M. R., Naylor, C. W. & Goldstein, A. L. (1992) Identification of immunoreactive forms ofThymosin 0tl in serum and supernatants by combining HPLC and RIA. Int. J. Immunopharm., 14, 1267-1278. Naylor, P. H., Naylor, C. W., Sasaki, D. & Mutchnick, M. G. (1994) Utilization of HPLC-ELISA to assess serum levels of thymosin ctl following subcutaneous administration to human subjects. J. Liquid Chromatoyr., 17, 3541-3551. Oates, K. K., McGinty, M. C. & Turner, T. (1988) Thymosin Qtl modulates proliferation of MCF-7 cells in culture. Med. Sci. Res., 16, 907-909. Sburlati, A. R., Manrow, R. E. & Berger, S. L. (1991) Prothymosin ct antisense oligomers inhibit myeloma cell division. Proc. Natl. Acad. Sci. USA, 88, 253-261. Sburlati, A. R., Rosa, A. D. L., Batey, D. W., Kurys, G. L., Manriwm, R. E., Pannell, L. K., Martin, B. M., Sheeley, D. M. & Berger, S. L. (1993) Phosphorylation of human and bovine prothymosin ct in vivo. Biochemistry, 32, 4587-4596. Schen, S. Y. (1987) Effect of thymosin ctl on Heptavax B among hemodialysis patients. Kidney Int., 31, 217-222. Schulof, R. S., Lloyd, J. J., Cleary, P. A., Palaszynski, S. R., Mai, D. A., Cox, J. W., Alabster, O. & Goldstein, A. U (1985) A randomized trial to evaluate the immunorestorative properties of synthetic thymosin ctl in patients with lung cancer. J. Biol. Resp. Mod., 4, 147-158. Schulof, R. S., Naylor, P. H., Sztein, M. & Goldstein, A. L. (1987) Thymic physiology and biochemistry. Adv. Clin. Chem., 26, 203-291. Smith, M. R., AI-Katib, A., Silverman, A., Szabo, P., Khilani, S., Kohler, W., Nath, R. & Mutchnick, M. G. (1993) Prothymosin ct gene expression correlates with proliferation, not differentiation of HL-60 cells. Blood, 82, 1127-1132. Szabo, P., Ehlieter, D., Whittington, E. & Weksler, M. F. (1992) Prothymosin alpha expression occurs during G1 in proliferating T or B lymphocytes. Biochem. Biophys. Res. Commun., 18, 953-959. Tsitsiloni, O. E., Stiakakis, J., Koutsellinis, A., Gogas, J., Markopuolos, C., Yialouris, P., Bekris, S., Panoussopoulos, D., Kiortsis, V., Voelter, W. & Haritos, A. A. (1993) Expression of ct-thymosins in human tissues in normal and abnormal growth. Proc. Natl. Acad. Sci. USA, 90, 9504-9507. Watts, J. D., Cary, P. D. & Crane-Robinson, C. (1989) Prothymosin alpha is a nuclear protein. FEBS Lett., 245, 17-20. Weller, F. E., Mutchnick, M. G., Keren, D. F., Goldstein, A. L. & Naylor, P. H. (1988) MicroELISA method for measurement of human serum thymosin otl. J. Immunol. Meth., 80, 45-53. Zalvido, J. B., Cancio, E., Alvarez, C. V., Magueiro, B. J. & Dominiquez, P. (1992) Prothymosin alpha mRNA levels are invariant throughout the cell cycle. J. Biol. Chem., 267, 8692-8695.