Phorbol Esters Induce Changes in Adenosine Deaminase, Furine

[CANCER RESEARCH 50. 2891-2894. May 15. 1990] Phorbol Esters Induce Changes in Adenosine Deaminase, Furine Nucleoside Phosphorylase, and Terminal Deoxynucleotidyl Transferase Messenger RNA Levels in Human Leukemic Cell Lines1

Vicente Madrid-Marina,2 Hector Martinez-Valdez,3 and Amos Cohen4

Division of Immunology/Rheumatology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8

ABSTRACT receptor molecules during gene rearrangements (12, 13). These observations led to the postulation that TdT, ADA, and PNP We have studied the expression of mRNA encoding adenosine deami- play a role in lymphocyte differentiation (14). nase (ADA; EC 3.5.4.4), purine nucleoside phosphorylase (PNP; EC We previously demonstrated that the ratio of ADA and PNP 2.4.2.1), and terminal deoxynucleotidyltransferase (TdT; EC 2.7.7.31) in different leukemic cell lines of B- and T-cell lineage. Incubation of activities plays a role in the regulation of intracellular deoxy- leukemic cells in the presence of the phorbol esters, 12-O-tetradecanoyl- nucleoside triphosphate pools in human thymocytes, which are phorbol-13-acetate or phorbol 12,13-dibutyrate, resulted in reduction of the substrates for TdT activity (15). It is therefore expected that ADA and TdT mRNA levels, while PNP mRNA levels increased under the expression of TdT, ADA and PNP mRNAs may be coor- the same treatment. The effect of TPA on the activity of these enzymes dinately regulated during T-cell antigen receptor gene re correlated well with its effects on their mRNA levels. TPA caused a 40% arrangement. Indeed, we have recently shown that the expres decrease in ADA and a 60% decrease in TdT enzyme activity, after 6 h sion of TdT, ADA, and PNP mRNAs are coordinately regulated of treatment. In contrast, PNP activity increased up to 200% after 12 h by phorbol esters in fresh isolated human thymocytes (16). The of incubation with the phorbol ester. phorbol ester TPA is an active modulator of mammalian cell The changes induced by the phorbol esters in the levels of mRNA of differentiation (17-19), a phenomenon that may be of relevance ADA, PNP, and TdT, and their enzyme activities in human leukemic cell lines mimic the changes in the activities of these enzymes in developing in developmentally lymphoid leukemic cells, arrested in early T-lymphocytes during differentiation in vivo, suggesting a role for protein stages of differentiation (20-23). Thus, the activities of these kinase C in the regulation of ADA, PNP, and TdT gene expression during enzymes can serve as markers of lymphoid cell differentiation. lymphoid cell differentiation. In order to investigate whether this pattern of regulation exists in undifferentiated malignant cells, we have studied the effect of phorbol esters on TdT, ADA, and PNP gene expression in INTRODUCTION human acute lymphoblastic leukemic cell lines of T- and pre- Immunodeficiency is often associated with inborn errors of B-cell lineage. purine metabolism. Deficiency of either one of the two enzymes which catalyze sequential reactions in purine catabolism, aden osine deaminase (EC 3.5.4.4) and purine nucleoside phospho MATERIALS AND METHODS rylase (EC 2.4.2.1), leads to severe combined immunodeficiency Cell Lines. A number of cell lines were used in this study: they include (1,2) and T-cell immunodeficiency (3, 4), respectively. ADA5 T-cell acute lymphoblastic leukemic cells; JURKAT, MOLT-3, CEM, catalyzes the conversion of adenosine and deoxyadenosine to and HSB-2, which were obtained from Dr. Hans-Michael Dosch (Di inosine and deoxyinosine; and PNP catalyzes the conversion of vision of Immunology, The Hospital for Sick Children, Toronto, On inosine, guanosine, and their respective deoxy derivatives to tario, Canada), and PEER, DND-41, and SKW-3 from Dr. T. W. Mak hypoxanthine (5). During the development of lymphocytes the (Ontario Cancer Institute, University of Toronto). Pre-B ALL cell activity of both enzymes change markedly. During T-lympho- lines, HYON, HOON, and NALM-6, were generously supplied by Dr. cyte differentiation ADA activity decreases and PNP activity Michelle Letarte (Division of Immunology, The Hospital for Sick increases (6-8). The enzyme activities in ALL patients resemble Children, Toronto). The cells were maintained in a logarithmic growth phase in RPMI 1640 medium supplemented with 10% fetal bovine those of immature thymocytes (9). serum, at 37Â°Cin a humidified incubator with 5% CO2 atmosphere. Terminal deoxynucleotidyl transferase (EC 2.7.7.31) cata Mycoplasma contamination was excluded by periodic microbiological lyzes the polymerization of DNA in the absence of a template. tests. Its activity is restricted to populations of normal immature Materials. [a-32P]dCTP (3000 Ci/mmol) was purchased from Du lymphocytes and malignant lymphocytes of immature pheno- Pont New England Nuclear (Lachine, Quebec, Canada). Phorbol esters: type (10, 11). The function of TdT is unclear; however, it has TPA, 4a-phorbol-12,13-didecanoate, 4j3-phorbol-12,13-didecanoate, been proposed that this enzyme plays an important role in the and phorbol-12,13-dibutyrate, sodium chloride, bovine serum albumin generation of diversity of immunoglobulin and T-cell antigen fraction V, sodium citrate, and salmon sperm DNA were obtained from Sigma Chemical Co. (St. Louis, MO). Stock solutions of phorbol esters Received 10/30/89; revised 2/2/90. (0.1 ITIM)were prepared in dimethyl sulfoxide (Fisher). Nylon transfer The costs of publication of this article were defrayed in part by the payment membranes (1.20 ^m.)were purchased from PALL BIODINE Ultrafine of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Filtration Co. (Glen Cove, NY). The TdT cDNA (24) was supplied by 1This work was supported by grants from the National Research Council of Dr. Bollimi (Uniformed Services University, Bethesda, MD), ADA Canada and the National Cancer Institute of Canada. cDNA (25) was provided by Dr. J. J. Hutton (Children's Hospital 2Supported by The Hospital for Sick Children Foundation. Postdoctoral Medical Center, Cincinnati, OH), and PNP cDNA (26) was obtained Fellowship. To whom requests for reprints should be addressed, at Division of Immunology/Rheumatology, Hospital for Sick Children, 555 University Avenue. from Dr. D. W. Martin, Jr. (Genentech, Inc., San Francisco, CA). Toronto. Ontario, Canada M5G 1X8. RNA Isolation and Northern Blot Analysis. The total RNA was 3Recipient of a Medical Research Council of Canada, Postdoctoral Fellowship. extracted in guanidinium isothiocyanate according to the method of 4 Recipient of the National Cancer Institute of Canada Scholarship. 5The abbreviations used are: ADA, adenosine deaminase; PNP, purine nucle Chomczynsky and Sacchi (27). RNA concentrations were measured with a double beam Beckman DU-40 spectrophotometer. Total RNA oside phosphorylase; ALL, acute lymphocytic leukemia: TdT, terminal deoxy nucleotidyl transferase; TPA. 12-O-tetradecanoylphorbol-13-acetate; cDNA, (10 fig) was electrophoresed under denaturing conditions on a 0.9% complementary DNA; PKC, protein kinase C; SSC. standard saline citrate. agarose and 0.66 M formaldehyde gel according to the method of 2891

Lehrach et al. (28). The gel was stained with 0.5 jug/m' of ethidium bromide to check the integrity of RNA. RNA was transferred onto nylon PLUS membranes as described by Thomas (29). Nylon mem o_g"eo branes were allowed to dry at room temperature and were baked for 1 h at 80Â°Cina vacuum oven. Blots were prehybridized for 12 h at 42Â°C in 50% formamide, 5x SSC (Ix SSC is 0.15 M NaCl-0.015 M sodium 0^Q_h-00Qu_LJJCL0HIO.ico.C'oEoCoo-Â».^Ã›Lr- citrate, pH 7.0); 5x Denhart's solution (Ix Denhart is 200 /Â¿MFicoll, 200 Â¿ig/mlpolyvinyl-pyrrolidone, and 200 pg/m\ bovine serum albumin fraction V); 25 mivi sodium phosphate, pH 6.5, 1.0% sodium dodecyl TdT * â€¢ft - sulfate, and 200 Mg/ml denatured salmon sperm DNA. Hybridizations were carried out by using the same prehybridization solution for 16 h by adding [Â«-"PjdCTP-labeled probes with the use of hexamer deoxy- oligonucleotides as random primers and the Klenow fragment of DNA polymerase I (Pharmacia Fine Chemicals, Uppsala, Sweden). The ADA probes were EcoR\ cDNA fragment encoding TdT (24), the ADA cDNA insert was excised with EcoRl and Ddel (25), and the PNP cDNA insert was excised with Pstl (26). Probes were isolated from low melting agarose gels. After hybridization blots were stringently washed for 2 h with three changes of 0.1 x SSC, 25 mM sodium phosphate, and 0.2% sodium dodecyl sulfate at 68Â°C,and were then autoradiographed at â€”70Â°Cbyusing a screen intensifier. Controls for equal amounts of PNP mRNA were performed by using the cDNA probe for the constitutively expressed gene ÃŸ-actin,which is a //in III fragment of a plasmid ob tained from the American Type Culture Collection (Rockville, MD). Slot Blot and Densitometry Scanning of mRNA Levels. After 4 h of incubation with 10 n\i TPA, the cell lines were harvested by centrifu- ÃŸ-ACTIN gation and total RNA was extracted as described above. RNA aliquots (10 fig, total) were spotted onto nitrocellulose, using a Schleicher & Schuell slot blotter apparatus and following manufacturers instructions. Nitrocellulose was allowed to dry at room temperature and then baked HYON for 2 h at 80Â°Cin a vacuum oven. Slot blots were prehybridized, Fig. 1. Northern blot analysis of TdT, ADA, and PNP mRNA in T-cells hybridized, washed, and autoradiographed as described for Northern treated with phorbol esters. Human leukemic T cells (PEER) were incubated for blots. Autoradiograms were scanned in a Beckman DU-40 spectropho- 4 h in the absence (Lane I) or presence of phorbol esters TPA (Lane 2), PDB (Lane 3 ), 4Â«-phorbol-12.13-didecanoate. (Lane 4), 4/3-phorbol-12,13-didecanoate tometer. The value of the cell sample incubated with no additions was (Lane 5). ionomycin (lono) (Lane 6), and the combination of TPA and Â¡onomycin normalized to 1.0 unit of relative absorbance, and other experimental (Lane 7). Total RNA was extracted as described in "Materials and Methods." Ten (jg of RNA/lane were electrophoresed in Ir; agarose gel, and transferred to points were calculated as the mRNA ratio (TPA/control). The results nylon membranes according to "Materials and Methods." The membranes were are representative of three or four experiments for each cell line. hybridized with the appropriate "P-labeled probe as indicated. The blots were Quantitation of Enzyme Activities and Protein. Specific activities were exposed for autoradiography for 18 h for PNP and /i-actin probes, and 72 h for determined in duplicate by using radiochemical methods as previously ADA and TdT probes. described for ADA (30), for PNP (31), and for TdT (32) in HYON cell line, and the results are representative of one or two experiments. Total cells is consistent with the activation of PKC, the effect of protein concentration was measured by the method of Bradford (33). inactive or active forms of phorbol esters on TdT, ADA, and PNP mRNA levels was tested. As shown in Figs. 1 and 2 the RESULTS phorbol ester with ÃŸconfiguration caused a decrease in the mRNA levels of TdT and ADA, and an increase in the mRNA The effects of phorbol esters on TdT, ADA, and PNP expres sion in human leukemic cells of T- and B-cell lineage were levels of PNP, while that with Â«configuration did not modify the mRNA levels of these enzymes. Thus, there was good analyzed by Northern blots. We chose to study HYON as a representative of B-cell lineage and PEER as a representative correlation between the structure of phorbol esters required for of T-cell lineage (Figs. 1 and 2). Incubation of these cell lines PKC activation and their effects produced on TdT, ADA, and with the phorbol esters TPA or phorbol-12,13-dibutyrate de PNP mRNA levels. To investigate whether the changes produced by phorbol creased the mRNA levels of ADA and TdT. In contrast, the mRNA levels of PNP were increased under the same conditions, esters on specific mRNA levels are followed by comparable in both B- and T-lymphoid cell lines. The quantitation of ADA, effects on enzyme activities, we compared the enzymatic activ ities of ADA, TdT, and PNP in cell-free extracts from the PNP, and TdT mRNA levels in a number of leukemic lines of B- and T-cell lineages treated with phorbol esters is summarized control and phorbol ester-treated cell cultures. Cultures con taining 1x10" cells/ml of the pre-B-cell line HYON were in Table 1. Incubation of the cell lines for 4 h in the presence or absence of the phorbol ester TPA (1.0 x 10~8 M), caused a incubated in the presence or absence of 10 nM TPA and samples reduction of ADA and TdT mRNA levels, while PNP mRNA were taken at 6, 12, 24, 50, and 80 h; the enzyme activity was levels increased in all leukemic cell lines tested. determined by using radiochemical analysis, as described in "Materials and Methods." The data in Fig. 3 show that after 6 Phorbol esters have a structure similar to diacylglycerol, the physiological activator of PKC, and activate PKC directly in h of treatment with TPA, there was a rapid fall in the enzymatic vivo and in vitro. There are several lines of evidence suggesting activity of both TdT and ADA, reaching 60 and 20% of control that PKC is the receptor for phorbol esters (17). Their effect values, respectively. PNP enzyme activity, on the other hand, depends on their chemical configuration, i.e., the ÃŸconfigura increased up to 200% after 6 h of incubation in the presence of tion is known to have high potency on PKC activation, whereas the phorbol ester. These results correlate well with the effects the a configuration is totally ineffective (17). In order to deter of phorbol esters on TdT, ADA, and PNP mRNA levels (Figs. mine whether the effect of phorbol esters on human leukemic 1 and 2). 2892

200-1 CO

LU LU 11 C < OÃ¹ CL CL o < o o. O Ã¼ CL Â¿St

TdT

ADA

40 TIME (hours) PNP Fig. 3. ADA, PNP, and TdT activities in TPA-treated cells. Cells (1 X IO6/ â€¢â€¢ ml) of the pre-B-cell line HYON were incubated with or without 10 niviTPA. At the indicated times, samples were taken and the enzyme activities were determined as described in "Materials and Methods." The enzyme activities are represented in percentage of change with respect to control. The basal levels of enzyme ÃŸ-ACTIN activities for the control cells were: 53.5 nmol/min/mg for ADA. 25.5 units/min/ ^W mg for TdT, and 20.0 nmol/min/mg for PNP.

levels increased (Table 1). These change in mRNA levels cor related well with changes in the enzymatic activities (Fig. 3). PEER Moreover, the changes induced by phorbol esters on the expres Fig. 2. Northern blot analysis of TdT, ADA. and PNP mRNA in pre-B-cells sion of these enzymes are consistent with variations of the TdT, treated with phorbol esters. Human leukemic pre-B-cells (HYON) were incubated ADA, and PNP enzyme activities during lymphocyte differen in the presence or absence of phorbol esters as is indicated in Fig. 1 legend. tiation in vivo (6-9). Our results on the regulation of ADA, TdT, and PNP expression by phorbol esters in leukemic cells, Table 1 Effects of TPA on ADA, TdT, and PNP mRNA levels in human leukemic cells reported in the present work, together with our previous studies Total RNA was extracted, slot blotted, and hybridized with appropriate radio in human thymocytes (16), strongly suggest a role for PKC active cDNA probes. Autoradiograms were scanned. The changes produced by activation in the coordinate regulation of TdT, ADA, and PNP TPA on ADA. PNP. and TdT mRNA levels in human leukemic cell lines are mRNA expression during lymphocyte differentiation. expressed in fold of changes with respect to control without treatment. TPA produces a decrease in the ADA and TdT mRNA levels, and an increase in the The vast majority of ALL cells express TdT enzymatic activ PNP mRNA levels in all the cell lines studied. ity, exclusively present in immature lymphocytes, and leukemic mRNA ratio TPA/control cells with immature phenotype only (10, 11). TdT is thought to participate in the synthesis of N-regions during gene re ADA PNP TdT arrangement of immunoglobulins and T-cell antigen receptor T-celllinesJURKATMOLT-3PEERCEMHSB-2DND-41SKW-3Pre-B-cell genes (12, 13). TdT activity is present in nondividing thymo cytes, and is active in the absence of de novo deoxynucleotide NE0.670.500.55NF.0.500.350.30synthesis operative during the S phase of the cycle cell (11, 12). We have previously shown that under these conditions, the ratio of the purine deoxynucleotide degrading enzymes ADA and PNP largely control the levels of dATP and dGTP pools needed for TdT activity (15), and our previous observations of linesHOONHYONNALM-60.350.450.420.530.400.430.330.500.300.4410.505.1810.401.671.80NDND2.252.942.600.50NE coordinate regulation of TdT, ADA, and PNP mRNA levels in freshly isolated thymocytes is consistent with this hypothesis (16). Here we have extended these observations to leukemic Â°NE, nonexpressed; ND, not done. cells of both B- and T-cell lineages. The results reported here further support the hypothesis that the expression of TdT, DISCUSSION ADA, and PNP are coordinately rtegulated during both B- and T-cell differentiation, indicating a common functional role of During the course of lymphocyte differentiation, the enzyme their enzyme activities during lymphocyte differentiation. activities of ADA and TdT decrease while the activity of PNP Taken together, these observations support the notion that ALL increases (6-9). Recently, we have shown that the expression cells of both B- and T-cell lineage are arrested at an immature of ADA, TdT, and PNP mRNA are coordinately regulated in stage of differentiation (34, 35), characterized by high ADA fresh isolated human thymocytes by phorbol esters. Phorbol and TdT levels, and that protein kinase C activation stimulates esters are active modulators of mammalian cell differentiation the differentiation of these leukemic cells further along their (17-20), a phenomenon that may be relevant in develop- respective lineages. mentally arrested leukemias arrested in early stages of differ entiation (20-23). We have shown in the present communica ACKNOWLEDGMENTS tion that incubation of human leukemic cells of T- and B-cell The authors are thankful to Drs. J. J. Hutton, D. W. Martin, Jr., lineage in the presence of various phorbol esters caused a and F. J. Mollimi for providing ADA, PNP, and TdT cDNA probes, decrease in TdT and ADA mRNA levels, while PNP mRNA respectively, and to Dr. Paul J. Doherty for reviewing the manuscript. 2893

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1990 American Association for Cancer Research. Phorbol Esters Induce Changes in Adenosine Deaminase, Purine Nucleoside Phosphorylase, and Terminal Deoxynucleotidyl Transferase Messenger RNA Levels in Human Leukemic Cell Lines

Vicente Madrid-Marina, Hector Martinez-Valdez and Amos Cohen

Cancer Res 1990;50:2891-2894.

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