THROMBOSIS RESEARCH 63; 557~561,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
COMMUNICATION
BRIEF
DNA-BINDING ACTIVITY OF THE PROTEINASE INHIBITOR TAPP C. Nastruzzil,
G. Feriottoz
and R. Gambari*2>3
1Department of Pharmaceutical Sciences, University of Ferrara, Italy. 21nstitute of Biological Chemistry, University of Ferrara, Italy. SBiotechnology Center, University of Ferrara, Italy. (Received 215.1991; accepted in revised form 10.6.1991 by Editor H.A. Vinazzer)
INTRODUCTION Control of the activity of proteinases is a very interesting topic in studies aimed at the identification of chemical compounds that could be used in the therapy of a variety of human diseases, including thrombosis (l4), inflammation (1, 5) and complement dependent immune reactions (l-4). aromatic polyamidines have been demonstrated to inhibit In this respect, activity of serine proteinases (1, 2, 4, 6) involved in thrombosis and such as factor Xa and thrombin. hemostasis, Accordingly, aromatic polyamidines exhibit a variety of biological effects, including inhibition of aggregation (4, 6). Therefore, a possible use of aromatic platelet polyamidines has been proposed in antiplatelet therapy for prevention and treatment of thromboembolic disorders (6), which are among the leading causes of morbidity and mortality in man. On the other hand, our research group has demonstrated that different compounds of this class inhibit tumor cell growth both “in vitro” (7) and “in vivo” (8). These effects were suggested to be related, in addition to inhibition of tumor proteinases, to a possible DNA-binding activity (9). In the present study we report the effects of the aromatic poly-amidine, tetra-p-amidinophenoxy-neo-pentane (TAPP) on the electrophoretic mobility of DNA fragments and on the interaction between nuclear DNAbinding proteins and target DNA sequences. To this aim we analysed the effects of TAPP on the binding of nuclear factor(s) to the octamer motif ATGCAAAT, which is present in different regulatory regions of eukaryotic and viral genes. Key Words:
f&matic
poly-amidine, tetra-p-amidinophenoxy-ne~qentane, 557
ENA.
558
DNA-BINDING ACTIVITY.
MATERIALS
AND METHODS
Chemicals. The aromatic polyamidine TAPP synthetized as previously reported (10). double stranded oligonucleotide, purchased
utilized in the present study was The sequence of the Ott-103 from Pharmacia (Sweden), is
5’-GCATGCCTGCAGGTCGACTCTAGAGGATGCTCCATGCA TCCTAGGTACGTTTACCTTCCCCGGGTACCGAGCTC-3’.
Unless otherwise (Switzerland).
stated
all
other
Vol. 63. No. 5
GGACGTACGGACGTCCAGCTGAGATC
chemicals
were
obtained
from
Fluka
Electrophoretic mobility shift assay. The electrophoretic mobility shift assay (shortly, gel retardation) was performed as described elsewhere (11). Nuclear extracts from K562 cell, cultured with standard condition in amedium (7), were prepared as reported (11) at protein concentration of l-5 mg/ml. Binding reactions were carried out in binding buffer (20 mM TrisHCl, pH 7.6, 50 mM KCl, 1 mM MgC12, 1 mM DTT, 0.2 mM EDTA, 0.01% Triton X-100,
5%
glycerol,
0.5
mM
spermidine),
in
the
presence
of
increasing
amounts of poly(dI:dC).poly(dI:dC) (Pharmacia, Sweden), 1 ug of nuclear extract proteins and 0.25 ng of labelled double stranded oligonucleotides, in a total volume of 25 u 1. Samples were electrophoresed on 10% polyacrylamide gels as described elsewhere (11). Gels were dried and exposed to X-Omat Kodak films at 80°C with intensifying screens.
RESULTS AND DISCUSSION Aromatic polyamidines exhibiting antiproteinase activity were recently proposed as potential drugs for the experimental treatment of thrombosis and emphysema (l-4). Among this class of compounds TAPP, was found to inhibit serine proteinases and platelet aggregation (4, 6, 10). TAPP was found to strongly bind In addition to these reported activities, DNA, as suggested by the retarded electrophoretic migration of Hind111 X DNA (FiglA) and by size exclusion of bacteriophage fragments chromatography of TAPP/DNA complexes (data not shown). As for other DNA-binding drugs (including distamycin and daunomycin) (12), we demonstrate that TAPP inhibits the interaction between DNA-binding proteins and specific DNA sequences, as shown in Fig.lB, which clearly mdicates that TAPP interferes with the binding of K562 nuclear factors to the ATGCAAAT octamer DNA motif. between nuclear factors and the Ott-103 The specificity of the interaction trier ‘(generating the OTF-1 band, shown in Fig.lB) was demonstrated by the following results. (a) Its intensity remains constant even after addition of 1 ug of poly(dI:dC).poly(dI:dC) and (b) no competition was detected after (up to 300 ng) of cold unrelated addition of increasing concentrations oligonucleotides (data not shown).
Vol. 63, No. 5
559
DNA-BINDING ACTIVITY...
kb u
OTF-1 b
TAPP FIG.1. A. Electrophoretic migration of ;1 electrophoresis, the DNA fragments have been concentrations of TAPP. Molecular weights of the left side of the panel. B. Effects of TAPP on the binding of nuclear sequence. The gel retardation experiment was
(uhh)
phage HindIll DNA fragments. Before incubated for 5 min with the indicated k phage DNA fragments are indicated on proteins to the octamer performed as described
ATGCAAAT elsewhere
target (I I).
1 ug of crude nuclear extracts from KS62 cells was mixed with the 32 P-labelled 103 bp target sequence containing the octamer ATTTGCAT motif, in a final volume of 25 ul in the presence of 400 ng of poly(dl:dC),poly(dI:dC) TAPP. The retarded band generated by the specific nuclear factors is arrowed. F = free Ott-103 mer.
and
the
interaction
indicated between
concentrations Ott-103
mer
of and
Therefore, in addition to short-term toxic effects on “in vivo” experimental animals (S), long-term effects (including neoplastic transformation) could be hypothesized for these class of compounds (as generally proposed for DNAbinding drugs) (12). Taken together these observations should introduce serious cautions in using these compounds in therapy, expecially in the treatment of disorders such as enphysema and thrombosis, for which alternative therapeutic approaches are available (5). Ackowledaements This research was Regione Veneto.
We thank Prof. R.Ferroni for the generous supported by AIRC and by Regione Emilia
gift of TAPP. Romagna and
REFERENCES 1.
GERATZ J. D., WHITMOR A. C., CHENG M. C. F., PIANTADOSI, C. Diamidino a-,Tir-diphenoxyalkanes. Structure-activity relationships the inhibition of thrombin, pancreatic kallikrein and trypsin. J Med Chem, 16, 970-975, 1973.
for
560
DNA-BINDING ACTIVITY..
Vol. 63, No. 5
2.
STURZEBECHER J., MARKWARDT F., WALSMANN P. Synthetic inhibitors of serine proteinases. XXIII. Inhibition of Factor Xa by diamidines. Thrombos Res, 17. 545-548, 1980.
3.
GLUSA E., BARTHEL W., MARKEARDT derivatives on human platelet function. (Stuttgard), 31, 172-178, 1974.
4.
FERRONI R., MENEGATII E., GUARNERI M., TADDEO U., ASCENZI P., AMICONI G. Aromatic amidines: inhibitory effect on purified plasma serine proteinases, blood coagulation and platelet aggregation. 11 Farmaco, 43, 5-13, 1988.
5.
TRAINOR D. A. Synthetic inhibitors of human neutrophil elastase. Trends in Pharmacol. Sciences (TIPS), 5, 303-307, 1987.
6.
MILAN1 L., FERRONI R., ZACCARINI M., TRANIELLO amidines with antiproteolitic activity inhibit platelet secretion. Thrombosis Res, 5_& 417-424, 1989.
7.
NASTRUZZI C., FERIOTTO G., BARBIERI R., FERRONI R., GUARNERI M., GAMBARI R. Differential effects of benzamidine derivatives on the expression of c-myc and HLA-DRa genes in a human B-lymphoid tumor cell line. Cancer Letters, 38, 297-305, 1988.
8.
BARTOLAZZI A., BARBIERI R., NASTRUZZI C., NATAL1 P.G., GAMBARI R. Antitumor Activity of the Proteinase Inhibitor Tetra-pamidinophenoxyneopentane in a Nude Mouse Model of Human Melanoma. In vivo, 3, 383-388, 1989.
9.
GAMBARI R., BARBIERI R., FERIOTTO G., SPANDIDOS D., NASTRUZZI Effects of the proteinase inhibitor tetra-p-amidinophonoxy neo pentane on in vitro adhesion and invasiveness of tumor cells. Anticancer Research, 10, 259-264, 1990.
10.
FERRONI R., MENEGATTI E., GUARNERI M., TADDEO U., BOLOGNESI M., ASCENZI P., AMICONI G. Aromatic tetra-amidines: synthesis of haloderivatives and their antiproteolytic activity. 11 Farmaco, 39, 901909, 1984.
11.
BARBIERI R., GIACOMINI P., VOLINIA S., NASTRUZZI C., MILE0 M., FERRINI U., SORIA M., BARRAI I., NATAL1 P.G., GAMBARI R. Human HLA-DRa gene: a rare oligonucleotide (GTATA) identifies an upstream sequence required for nuclear protein binding. FEBS Letters, 268, 51-54, 1990.
F. The influence of benzamidine Thrombos Diathes Haemorrh
S. Aromatic aggregation
tetraand
C.
Vol. 63. No. 5
12.
DNA-BINDING ACTIVITY...
GAMBARI R., GIACOMINI P., ARCAMONE F. DNA-binding drugs inhibiting the interaction between nuclear factors and target DNA sequences. J Cancer Res and Clin Oncol, 116, 1107, 1990.
561
COMMUNICATION
BRIEF
DNA-BINDING ACTIVITY OF THE PROTEINASE INHIBITOR TAPP C. Nastruzzil,
G. Feriottoz
and R. Gambari*2>3
1Department of Pharmaceutical Sciences, University of Ferrara, Italy. 21nstitute of Biological Chemistry, University of Ferrara, Italy. SBiotechnology Center, University of Ferrara, Italy. (Received 215.1991; accepted in revised form 10.6.1991 by Editor H.A. Vinazzer)
INTRODUCTION Control of the activity of proteinases is a very interesting topic in studies aimed at the identification of chemical compounds that could be used in the therapy of a variety of human diseases, including thrombosis (l4), inflammation (1, 5) and complement dependent immune reactions (l-4). aromatic polyamidines have been demonstrated to inhibit In this respect, activity of serine proteinases (1, 2, 4, 6) involved in thrombosis and such as factor Xa and thrombin. hemostasis, Accordingly, aromatic polyamidines exhibit a variety of biological effects, including inhibition of aggregation (4, 6). Therefore, a possible use of aromatic platelet polyamidines has been proposed in antiplatelet therapy for prevention and treatment of thromboembolic disorders (6), which are among the leading causes of morbidity and mortality in man. On the other hand, our research group has demonstrated that different compounds of this class inhibit tumor cell growth both “in vitro” (7) and “in vivo” (8). These effects were suggested to be related, in addition to inhibition of tumor proteinases, to a possible DNA-binding activity (9). In the present study we report the effects of the aromatic poly-amidine, tetra-p-amidinophenoxy-neo-pentane (TAPP) on the electrophoretic mobility of DNA fragments and on the interaction between nuclear DNAbinding proteins and target DNA sequences. To this aim we analysed the effects of TAPP on the binding of nuclear factor(s) to the octamer motif ATGCAAAT, which is present in different regulatory regions of eukaryotic and viral genes. Key Words:
f&matic
poly-amidine, tetra-p-amidinophenoxy-ne~qentane, 557
ENA.
558
DNA-BINDING ACTIVITY.
MATERIALS
AND METHODS
Chemicals. The aromatic polyamidine TAPP synthetized as previously reported (10). double stranded oligonucleotide, purchased
utilized in the present study was The sequence of the Ott-103 from Pharmacia (Sweden), is
5’-GCATGCCTGCAGGTCGACTCTAGAGGATGCTCCATGCA TCCTAGGTACGTTTACCTTCCCCGGGTACCGAGCTC-3’.
Unless otherwise (Switzerland).
stated
all
other
Vol. 63. No. 5
GGACGTACGGACGTCCAGCTGAGATC
chemicals
were
obtained
from
Fluka
Electrophoretic mobility shift assay. The electrophoretic mobility shift assay (shortly, gel retardation) was performed as described elsewhere (11). Nuclear extracts from K562 cell, cultured with standard condition in amedium (7), were prepared as reported (11) at protein concentration of l-5 mg/ml. Binding reactions were carried out in binding buffer (20 mM TrisHCl, pH 7.6, 50 mM KCl, 1 mM MgC12, 1 mM DTT, 0.2 mM EDTA, 0.01% Triton X-100,
5%
glycerol,
0.5
mM
spermidine),
in
the
presence
of
increasing
amounts of poly(dI:dC).poly(dI:dC) (Pharmacia, Sweden), 1 ug of nuclear extract proteins and 0.25 ng of labelled double stranded oligonucleotides, in a total volume of 25 u 1. Samples were electrophoresed on 10% polyacrylamide gels as described elsewhere (11). Gels were dried and exposed to X-Omat Kodak films at 80°C with intensifying screens.
RESULTS AND DISCUSSION Aromatic polyamidines exhibiting antiproteinase activity were recently proposed as potential drugs for the experimental treatment of thrombosis and emphysema (l-4). Among this class of compounds TAPP, was found to inhibit serine proteinases and platelet aggregation (4, 6, 10). TAPP was found to strongly bind In addition to these reported activities, DNA, as suggested by the retarded electrophoretic migration of Hind111 X DNA (FiglA) and by size exclusion of bacteriophage fragments chromatography of TAPP/DNA complexes (data not shown). As for other DNA-binding drugs (including distamycin and daunomycin) (12), we demonstrate that TAPP inhibits the interaction between DNA-binding proteins and specific DNA sequences, as shown in Fig.lB, which clearly mdicates that TAPP interferes with the binding of K562 nuclear factors to the ATGCAAAT octamer DNA motif. between nuclear factors and the Ott-103 The specificity of the interaction trier ‘(generating the OTF-1 band, shown in Fig.lB) was demonstrated by the following results. (a) Its intensity remains constant even after addition of 1 ug of poly(dI:dC).poly(dI:dC) and (b) no competition was detected after (up to 300 ng) of cold unrelated addition of increasing concentrations oligonucleotides (data not shown).
Vol. 63, No. 5
559
DNA-BINDING ACTIVITY...
kb u
OTF-1 b
TAPP FIG.1. A. Electrophoretic migration of ;1 electrophoresis, the DNA fragments have been concentrations of TAPP. Molecular weights of the left side of the panel. B. Effects of TAPP on the binding of nuclear sequence. The gel retardation experiment was
(uhh)
phage HindIll DNA fragments. Before incubated for 5 min with the indicated k phage DNA fragments are indicated on proteins to the octamer performed as described
ATGCAAAT elsewhere
target (I I).
1 ug of crude nuclear extracts from KS62 cells was mixed with the 32 P-labelled 103 bp target sequence containing the octamer ATTTGCAT motif, in a final volume of 25 ul in the presence of 400 ng of poly(dl:dC),poly(dI:dC) TAPP. The retarded band generated by the specific nuclear factors is arrowed. F = free Ott-103 mer.
and
the
interaction
indicated between
concentrations Ott-103
mer
of and
Therefore, in addition to short-term toxic effects on “in vivo” experimental animals (S), long-term effects (including neoplastic transformation) could be hypothesized for these class of compounds (as generally proposed for DNAbinding drugs) (12). Taken together these observations should introduce serious cautions in using these compounds in therapy, expecially in the treatment of disorders such as enphysema and thrombosis, for which alternative therapeutic approaches are available (5). Ackowledaements This research was Regione Veneto.
We thank Prof. R.Ferroni for the generous supported by AIRC and by Regione Emilia
gift of TAPP. Romagna and
REFERENCES 1.
GERATZ J. D., WHITMOR A. C., CHENG M. C. F., PIANTADOSI, C. Diamidino a-,Tir-diphenoxyalkanes. Structure-activity relationships the inhibition of thrombin, pancreatic kallikrein and trypsin. J Med Chem, 16, 970-975, 1973.
for
560
DNA-BINDING ACTIVITY..
Vol. 63, No. 5
2.
STURZEBECHER J., MARKWARDT F., WALSMANN P. Synthetic inhibitors of serine proteinases. XXIII. Inhibition of Factor Xa by diamidines. Thrombos Res, 17. 545-548, 1980.
3.
GLUSA E., BARTHEL W., MARKEARDT derivatives on human platelet function. (Stuttgard), 31, 172-178, 1974.
4.
FERRONI R., MENEGATII E., GUARNERI M., TADDEO U., ASCENZI P., AMICONI G. Aromatic amidines: inhibitory effect on purified plasma serine proteinases, blood coagulation and platelet aggregation. 11 Farmaco, 43, 5-13, 1988.
5.
TRAINOR D. A. Synthetic inhibitors of human neutrophil elastase. Trends in Pharmacol. Sciences (TIPS), 5, 303-307, 1987.
6.
MILAN1 L., FERRONI R., ZACCARINI M., TRANIELLO amidines with antiproteolitic activity inhibit platelet secretion. Thrombosis Res, 5_& 417-424, 1989.
7.
NASTRUZZI C., FERIOTTO G., BARBIERI R., FERRONI R., GUARNERI M., GAMBARI R. Differential effects of benzamidine derivatives on the expression of c-myc and HLA-DRa genes in a human B-lymphoid tumor cell line. Cancer Letters, 38, 297-305, 1988.
8.
BARTOLAZZI A., BARBIERI R., NASTRUZZI C., NATAL1 P.G., GAMBARI R. Antitumor Activity of the Proteinase Inhibitor Tetra-pamidinophenoxyneopentane in a Nude Mouse Model of Human Melanoma. In vivo, 3, 383-388, 1989.
9.
GAMBARI R., BARBIERI R., FERIOTTO G., SPANDIDOS D., NASTRUZZI Effects of the proteinase inhibitor tetra-p-amidinophonoxy neo pentane on in vitro adhesion and invasiveness of tumor cells. Anticancer Research, 10, 259-264, 1990.
10.
FERRONI R., MENEGATTI E., GUARNERI M., TADDEO U., BOLOGNESI M., ASCENZI P., AMICONI G. Aromatic tetra-amidines: synthesis of haloderivatives and their antiproteolytic activity. 11 Farmaco, 39, 901909, 1984.
11.
BARBIERI R., GIACOMINI P., VOLINIA S., NASTRUZZI C., MILE0 M., FERRINI U., SORIA M., BARRAI I., NATAL1 P.G., GAMBARI R. Human HLA-DRa gene: a rare oligonucleotide (GTATA) identifies an upstream sequence required for nuclear protein binding. FEBS Letters, 268, 51-54, 1990.
F. The influence of benzamidine Thrombos Diathes Haemorrh
S. Aromatic aggregation
tetraand
C.
Vol. 63. No. 5
12.
DNA-BINDING ACTIVITY...
GAMBARI R., GIACOMINI P., ARCAMONE F. DNA-binding drugs inhibiting the interaction between nuclear factors and target DNA sequences. J Cancer Res and Clin Oncol, 116, 1107, 1990.
561
