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Stem Cells, Vol. 17, No. 2, 100-106, March 1999
© 1999 AlphaMed Press

In Vitro Effect of Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) Analogs Resistant to Angiotensin I-Converting Enzyme on Hematopoietic Stem Cell and Progenitor Cell Proliferation

Sandrine Gaudrona, Catherine Grillona, Josiane Thierrya, Andrew Richesb, Pieter K. Wierengac, Joanna Wdzieczak-Bakalaa

a Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France;
b School of Biological and Medical Sciences, University of St Andrews, St Andrews, Scotland, UK;
c Department of Radiobiology, University of Groningen, Groningen, The Netherlands

Key Words. AcSDKP • Proliferation inhibitor • AcSDKP analogs • Pseudopeptides • Hematopoietic stem cell • ACE

Dr. Catherine Grillon, Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette Cedex, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP), an inhibitor of hematopoietic stem cell proliferation, is known to reduce in vivo the damage resulting from treatment with chemotherapeutic agents or ionizing radiation on the stem cell compartment. Recently, AcSDKP has been shown to be a physiological substrate of the N-active site of angiotensin I-converting enzyme (ACE). Four analogs of the tetrapeptide expressing a high stability towards ACE degradation in vitro have been synthesized in order to provide new molecules likely to improve the myeloprotection displayed by AcSDKP. These analogs are three pseudopeptides with a modified peptidic bond, Ac-Ser{Psi}(CH2-NH)Asp-Lys-Pro, Ac-Ser-Asp{Psi}(CH2-NH)Lys-Pro, Ac-Ser-Asp-Lys{Psi}(CH2-N)Pro, and one C-terminus modified peptide (AcSDKP-NH2). We report here that these analogs reduce in vitro the proportion of murine colony-forming units-granulocyte/macrophage in S-phase and inhibit the entry into cycle of high proliferative potential colony-forming cells. The efficacy of AcSDKP analogs in preventing in vitro primitive hematopoietic stem cells from entering into cycle suggests that these molecules could be new candidates for the powerful inhibition of hematopoietic stem and progenitor cell proliferation in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hematopoiesis is under the control of a set of positive and negative factors which are recognized as a new class of biological modifiers of chemo- and radiotherapy response in vitro and in vivo [1-3]. Thus, the interest of inhibitors of hematopoiesis lies not only in their regulatory function but also in their potential clinical use as marrow protectors against the toxicity of anticancer treatments [4, 5].

The tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) isolated from bone marrow has been identified as one of the inhibitors of hematopoietic stem cell proliferation [6]. In vitro, AcSDKP prevents the proliferative response of purified human CD34+ cells to the action of growth factors at nanomolar concentrations [7] as well as the proliferative response of primitive hematopoietic cells, such as the murine high proliferative potential colony-forming cells (HPP-CFC) triggered into cell cycle by stimulators [8]. Further, it inhibits clonal growth and the recruitment into S-phase of human and murine early progenitors [9, 10]. However, AcSDKP has no effect on the proliferative status of leukemic progenitors [11, 12], and therefore it may selectively prevent the progress of normal stem cells into cycle. The ability of AcSDKP to maintain the quiescence of primitive hematopoietic cells is considered to be responsible for the in vitro protection of human or murine progenitors from the toxicities of zidovudine [13], mafosfamide (ASTA Z) [14], phototherapy [15], and hyperthermy [16-18].

In vivo, AcSDKP prevents murine stem cells and early progenitors from entering into S-phase following cytotoxic drug administration [6, 19, 20] or irradiation [21, 22]. It has also been shown that AcSDKP given to normal mice or monkeys reduces significantly the percentage of cycling bone marrow progenitors [23]. These activities allow protection of the stem cell compartment from the cytotoxicity of anticancer drugs and consequently lead to an increase in survival of animals when AcSDKP administration is associated with a chemotherapeutic regimen [19, 24, 25]. All of these biological properties of AcSDKP as well as the absence of antiproliferative activity of this tetrapeptide on leukemic cells suggest possible therapeutic applications for AcSDKP in vivo as an efficient hemoprotective agent during repeated and intensive chemo- and radiotherapeutic treatment, and in vitro as an adjuvant to purging methods. The results of phase I-II clinical trials have already demonstrated a reduced period of neutropenia in cancer patients receiving AcSDKP and either cytosine arabinoside or ifosfamide [26].

Studies on the catabolism of AcSDKP revealed that, in human plasma, the soluble angiotensin-I converting enzyme (ACE) is responsible in vitro for the limiting step of AcSDKP hydrolysis [27]. ACE (peptidyl dipeptidase A, kininase II, EC 3.4.15.1) is a zinc-dipeptidyl carboxypeptidase whose major physiological functions are the conversion of angiotensin-I into the potent vasoconstrictor angiotensin-II and the inactivation of the vasodilator bradykinin [28]. These activities explain the predominant role of ACE in the regulation of blood pressure. The somatic form of ACE was shown to contain two functional N- and C-terminal catalytic domains [29]. In vitro, studies developed first with the wild-type and the two full-length mutants of ACE expressing only one active site and next with natural single-domain germinal and ileal ACE revealed that the N-active domain of ACE was mainly involved in the degradation of AcSDKP [30, 31]. Furthermore, the catalytic efficiency of the N-active mutant ACE was similar to that of the wild-type, suggesting that AcSDKP could be the physiological substrate of the N-active site of ACE. The results of in vivo studies carried out in humans confirmed the previous observations, showing that ACE was the major enzyme involved in vivo in the catabolism of the tetrapeptide [32]. In fact, AcSDKP represents the first physiological substrate specific to the N-active site of ACE.

The investigations of AcSDKP pharmacokinetics in healthy volunteers have shown a quick elimination phase for intravenously infused tetrapeptide, with a mean half-life of 4.5 min [33]. As the in vivo half-life is extremely short, this will severely compromise its potential clinical use as a myeloprotective agent in anticancer treatment. In fact, to elicit a proper therapeutic response it is essential to maintain a pharmacologically active peptide concentration in the blood for a prolonged period. Therefore, we were prompted to develop new AcSDKP-derived molecules exhibiting a higher stability in the blood; such molecules have been designed and synthesized recently [34].

In the present paper, we report the biological efficacy of four analogs of AcSDKP resistant to ACE action, three pseudopeptides and one C-terminus modified peptide. The use of such active AcSDKP analogs able to inhibit the proliferation of primitive hematopoietic cells could offer new opportunities for improving the myeloprotection displayed by AcSDKP in the radio- or chemotherapeutic management of cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptide and Analogs
The synthetic AcSDKP was kindly provided by IPSEN-Biotech (Paris, France). The three pseudopeptides Ac-Ser{Psi}(CH2-NH)Asp-Lys-Pro (S{Psi}), Ac-Ser-Asp{Psi}(CH2-NH) Lys-Pro (D{Psi}), and Ac-Ser-Asp-Lys{Psi}(CH2-N)Pro (K{Psi}); the C-terminus modified peptide Ac-Ser-Asp-Lys-Pro-NH2 (AcSDKP-NH2); and the structurally distinct analog of the tetrapeptide, AcSDDKP, where L-Asp was replaced by its optical D-isomer, were synthesized in our laboratory as previously described [34, 35]. The structures of the studied compounds are presented in Figure 1.



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  		Figure 1. Primary structure of AcSDKP and its analogs.

 
Hematopoietic Cells
Bone marrow from normal 9- to 11-week-old CBA/J mice was used as the source of hematopoietic bone marrow cells (BMC). BMC were obtained by flushing the content of tibias and femurs with Dulbecco's medium or {alpha}-medium (GIBCO-Life Technology; Cergy Pontoise, France).

Colony-Forming Unit-Granulocyte/Macrophage (CFU-GM) Assay
The assay was carried out according to slightly modified procedures described previously by Wierenga and Konings [16]. BMC were incubated in {alpha}-medium (3 x 106 cells/ml) for 7 h at 37°C in a 5% CO2 humidified atmosphere, either with culture medium as control or with AcSDKP or analogs (2 x 10–9 M final concentration). AcSDDKP was used systematically in all assays as a negative control. Hydroxyurea ([HU], 200 µg/ml final concentration) or culture medium was then added to a duplicate culture for a 1-h incubation. Cells were washed twice and plated out for the colony-forming assay. One ml of complete medium ({alpha}-medium containing 30% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin) supplemented with 0.8% methylcellulose, 10% pokeweed mitogen-stimulated spleen-conditioned medium as source of growth factors, and 4 x 104 BMC were aliquoted in 35-mm polystyrene culture dishes. Quadruplicate cultures were incubated for eight days at 37°C in a fully humidified atmosphere with 5% CO2. The colonies were counted using an inverted microscope. Groups of more than 50 cells were counted as colonies. For each condition, the proportion of cells in S-phase (mean ± SE) was estimated from the difference in the number of colonies between cells treated with HU and those treated without it. The whole experiment was repeated three times. The effect of the addition of AcSDKP or analogs versus control was evaluated through an analysis of variance performed on the proportion of cells in S-phase, taking into account the matching of data arising from the same experiment, the intra- and inter-experiment variations of the data, and the number of comparisons performed through Dunnett's t-test.

HPP-CFC Assay
This assay was carried out according to the procedure described by Robinson et al. [8]. BMC were incubated in Dulbecco's medium (5 x 106 cells/ml) for 2 h at 37°C, either with culture medium as control or with 3-h conditioned medium of BMC from mice sublethally irradiated seven days before as a crude source of stimulators. AcSDKP or analogs (2 x 10–9 M final concentration, or 10–13 to 10–5 M for the dose-response study) or medium, as stimulation control, were added at the beginning of the incubation. Cytosine arabinoside ([AraC], 50 µg/ml final concentration) or culture medium was then added for a 1-h incubation. Cells were washed twice prior to HPP-CFC assay. Two ml of complete medium (Dulbecco's medium containing 20% horse serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin) supplemented with 10% conditioned medium from the WEHI 3B myelomonocytic leukemic cell line, 10% conditioned medium from L929 fibroblast cell line as source of growth factors, and 0.5% melted agar (Bactoagar, Difco; Detroit, MI) were aliquoted into 55 mm diameter non-tissue culture grade plastic petri dishes as the underlayer. Two ml of complete medium supplemented with 0.3% melted agar and containing 3 x 104 BMC/ml were then aliquoted over the prepared underlayers. Quadruplicate cultures were incubated for 14 days at 37°C in a fully humidified atmosphere with 5% CO2. Twelve hours before the end of the culture, cells were stained by adding 1 ml of a 1 mg/ml 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride ([INT], Sigma; Saint Quentin Fallavier, France) solution in saline. HPP-CFC macroscopic colonies defined as those in excess of 1 mm were scored. For each condition, the proportion of cells in S-phase (mean ± SE) was estimated from the difference in the number of colonies between cells treated with AraC and those treated without it. The whole experiment was repeated three times. The effect of stimulating medium versus control and of the addition of AcSDKP or analogs versus stimulating medium alone was evaluated through an analysis of variance performed on the proportion of cells in S-phase, using Dunnett's t-test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effect of AcSDKP analogs on the proliferative status of the murine committed progenitors, CFU-GM, was first evaluated. As shown in Figure 2, in normal bone marrow, 34.1 ± 0.6% of the CFU-GM appear to be in S-phase. This value was significantly decreased to 11.9 ± 0.6 % (p < 0.01) following an 8-h exposure of BMC to 2 x 10–9 M AcSDKP. When BMC were incubated in the presence of 2 x 10–9 M pseudopeptides S{Psi}, D{Psi}, or K{Psi}, or of the C-terminus modified peptide AcSDKP-NH2, the proportion of CFU-GM in S-phase was also significantly decreased to 9.8 ± 1.8%, 7.3 ± 8.2%, 9.9 ± 6.3%, and 13.9 ± 4.4%, respectively (p < 0.01). Conversely, the isomer AcSDDKP, used currently as a negative control, was unable to modify the percentage of CFU-GM in S-phase (36.2 ± 3.1%). It should be stressed that the total number of cultured CFU-GM remained constant in the control and all the cultures whatever the peptide used, indicating a lack of toxicity of all studied molecules (Table 1).



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  		Figure 2. Effect of AcSDKP analogs on the proportion of CFU-GM in S-phase. BMC were incubated with control medium (control cells), with AcSDKP or the analog 2 x 10–9 M. Results are expressed as the percentage of CFU-GM in S-phase (mean <± SE from three separate experiments). ** p < 0.01 in comparison with control cells; o p < 0.01, NS = nonsignificant in comparison with AcSDKP.

 

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  		Table 1. Effect of AcSDKP and analogs on the number of HPP-CFC and CFU-GM colonies
 
The biological activity of AcSDKP analogs was then investigated using in vitro assay of the primitive hematopoietic cells HPP-CFC. As shown in Figure 3, the percentage of HPP-CFC population in S-phase in nontreated BMC was lower than 4%, indicating that the HPP-CFC population was initially quiescent. Following a 2-h incubation of BMC with medium conditioned by sublethally x-irradiated regenerating femoral marrow as a crude source of stimulators, 32.9 ± 1.9% of HPP-CFC were triggered into cycle. In the presence of 2 x 10–9 M AcSDKP, pseudopeptides, or C-terminus modified peptide, the proportion of cells recruited into cycle was markedly diminished. The percentage of HPP-CFC in S-phase was 12.8 ± 2.6%, 11.8 ± 4.8%, 11.5 ± 8.5%, and 15.1 ± 7.7%, respectively for AcSDKP, S{Psi}, D{Psi}, and K{Psi}, and 14.7 ± 2.1% for AcSDKP-NH2. Conversely, the isomer AcSDDKP was shown to have no inhibitory effect on HPP-CFC entry into cycle (26.6 ± 1.6% of HPP-CFC in S-phase). Moreover, the incubation of BMC with AcSDKP or analogs induces no change in the number of HPP-CFC, showing a lack of cytotoxicity of these molecules (Table 1).



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  		Figure 3. Effect of AcSDKP analogs on the entry into S-phase of HPP-CFC. BMC were incubated with control medium (control cells), with stimulating medium in the absence (stimulated cells) or in the presence of AcSDKP or analog 2 x 10–9 M. Results are expressed as the percentage of HPP-CFC in S-phase (mean ± SE from three separate experiments). ** p < 0.01, * p < 0.05 in comparison with stimulated cells; o p < 0.01, NS = nonsignificant in comparison with AcSDKP.

 
In order to compare the in vitro inhibitory potency of the pseudopeptide D{Psi} and AcSDKP on HPP-CFC entry into S-phase, a dose-response study was undertaken. As shown in Figure 4, the largest inhibitory effect was observed at 10–9 M for both D{Psi} and AcSDKP (12.0 ± 0.7% and 12.8 ± 0.9% of HPP-CFC in S-phase, respectively). The inhibitory activities of D{Psi} and AcSDKP were not significantly different except at the concentration of 10–5 M (p < 0.05).



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  		Figure 4. Comparative dose-effect of D {Psi} and AcSDKP on the entry into S-phase of HPP-CFC. BMC were incubated with control medium (CC: control cells), with stimulating medium in the absence (SC: stimulated cells) or in the presence of AcSDKP or D {Psi} 10–13 to 10–5 M. Results are expressed as the percentage of HPP-CFC in S-phase (mean ± SE from three separate experiments). ** p < 0.01, * p < 0.05 in comparison with stimulated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The tetrapeptide AcSDKP belongs to the family of negative regulators of hematopoiesis. It inhibits in vivo and in vitro the entry into S-phase of human and murine stem cells and committed progenitors. In vivo studies in mice demonstrated that AcSDKP acts on hematopoietic stem cells at the transition between G0 and G1 or in the early G1-phase of the cell cycle [36]. Such proliferative arrest decreases the sensitivity of responding cells to the toxicity of various anticancer treatments. Recently, it has been reported that the dose and time schedule of AcSDKP administration are critical in obtaining the optimal myeloprotective effect against toxicity of chemotherapy [20, 25] and radiotherapy [21]. The concentration of exogenous AcSDKP—and thus the duration of action of the peptide and the consequent range of time available to maintain hematopoietic target cells in G0—depends in part on the catabolism of the administered tetrapeptide. Indeed, AcSDKP, a circulating natural peptide, is continuously degraded by ACE via its dipeptidyl carboxypeptidase activity [30, 32]. Since in vivo AcSDKP appeared to have a very short half-life [33], it was of interest to design and develop new molecules resistant to proteolysis which were able to mimic the biological activity of AcSDKP. In fact, it is highly probable that such analogs could improve the therapeutic index of AcSDKP and consequently could be more suitable for long-term use in cancer patients. It should be considered, however, that the limited duration of AcSDKP reported in healthy individuals is not the only factor interfering with the efficacy of the tetrapeptide given during cancer therapy. It was recently demonstrated in mice that both administration of cytotoxic drugs [37] and irradiation [38] are followed by a transient increase of ACE activity and a consequent accelerated catabolism of AcSDKP. Moreover, significant changes in plasma AcSDKP levels have been reported in patients with acute myeloid leukemia undergoing chemotherapy [39]. In order to minimize the consequences of AcSDKP degradation in vivo, we have prepared various analogs expected to be resistant to ACE activity. In the three pseudopeptides, one of the peptide bonds has been replaced by an aminomethylene group: {Psi} (CH2NH). On the other hand, AcSDKP C-terminus has been modified by amidation. All of these structural modifications were shown to improve the stability of these molecules towards proteolysis in plasma when incubated in vitro [34]. It was suggested that this increased resistance towards the degradation by ACE could be due to the weak affinity of the analogs for the active site of the enzyme. These findings are in agreement with the results of studies concerning enkephalin analogs displaying different modified bonds which were also reported to be resistant to degradation in human plasma [40]. Furthermore, it will also be necessary to ascertain whether other AcSDKP degradative pathways exist in vivo, which seems unlikely, as ACE—the major enzyme responsible for in vitro degradation of AcSDKP—and ACE inhibitors have dramatic effects on the catabolism of AcSDKP both in vitro and in vivo.

The studies presented in this paper were developed to provide data on the biological activity of molecules expressing a higher stability towards the degradative potency of ACE. Two in vitro clonogenic assays were used to investigate the ability of the analogs to prevent the progress into cell cycle of two different types of bone marrow cells, HPP-CFC and CFU-GM. In fact, these cell populations were shown previously to respond to AcSDKP [8, 16]. We report here that all tested compounds retain full biological activity as inhibitors of the entry into S-phase of HPP-CFC and CFU-GM and that these effects are not mediated by a cytotoxic activity on the hematopoietic progenitors and stem cells. The total lack of toxicity in vitro of these highly stable molecules does not exclude the possibility that they may induce side effects in vivo. However, it seems unlikely that low doses of compounds derived from AcSDKP, which is completely devoid of any toxicity in vivo, could induce any side effects under the same conditions. But this will need to be examined in detail.

The present results indicate clearly that the peptide backbone is not essential for the conservation of the biological activity of AcSDKP. The structural modifications consisting of the replacement of a peptide bond by a CH2-NH group do not modify the inhibitory effect of AcSDKP, indicating correct spacing of the amino acid side chains. Indeed, it has been previously shown that the side chains of lysine and aspartic acid are important for the expression of AcSDKP activity [35, 41], suggesting their implication in the interaction between AcSDKP and its yet unidentified molecular target. The structural feature of the C-terminus modified peptide was not critical for its biological activity. We also demonstrated that decarboxylation of the C-terminal proline (Acetyl-Ser-Asp-Lys-Pyrrolidide) did not lead to any loss of the inhibitory activity (results not shown). These data are in line with the results of a structure-function relationship study which has shown that the tripeptide sequence SDK was essential for AcSDKP biological activity. In fact, the deletion of proline from the tetrapeptide sequence was not detrimental to the activity of the resulting tripeptide [35, 41]. The statistical analysis of the results has shown that the four AcSDKP analogs were able to inhibit the recruitment into DNA synthesis of the target cells with the same efficacy as AcSDKP. The comparative dose-response studies over a large concentration range (varying from 10–5 M to 10–13 M) of AcSDKP and D{Psi} confirmed similar potency for decreasing the proportion of HPP-CFC in S-phase of the parent peptide and its analog. The most important effect was obtained at 10–9 M for both compounds. The bell-shaped dose response demonstrated for D{Psi} has also been reported for AcSDKP [7, 9], which we cannot observe here, as the range of doses was probably not wide enough. The fact that D{Psi} acts only at 10–7 and 10–9 M may reflect a lower affinity than AcSDKP, probably linked to the modification of its structure. However, this does not permit any assumptions about its in vivo myeloprotective activity.

In order to further assess the therapeutic potential of these active analogs, in vivo studies which aim to evaluate their ability to reduce the acute hematotoxicity of anticancer drugs will be undertaken. It will be important to confirm whether these active analogs also exhibit a differential effect on tumor and leukemic cells, as has been demonstrated with AcSDKP [11, 12]. Optimization of the myeloprotective effects of AcSDKP analogs resistant to proteolysis could provide an additional prospect for cancer patients to maintain their hematopoietic capacity following intensive chemo- or radiotherapy. The development of AcSDKP analogs with useful therapeutic properties may require additional synthetic work in order to improve pharmacokinetic properties. The present active molecules offer excellent lead structures for this type of research.


   Acknowledgments
 
The authors thank Dr. J.Y. Mary for his valuable help in statistical analysis, Ipsen-Biotech for its financial support, and Dr. E. Deschamps de Paillette for her constant encouragement.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication December 21, 1998.



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