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Author for correspondence (Fax: +55 11 30917420; E-mail: gpadilla@icb.usp.br). Received 4 July 2002; Revisions requested 17 July 2002; Revisions received ...
Biotechnology Letters 24: 1807–1813, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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A rapid and sensitive method for the screening of DNA intercalating antibiotics Renata Ligia A. Furlan1 , Leandro M. Garrido2 , Gabriela Brumatti3 , Gustavo P. AmaranteMendes3 , Renata A. Martins4, Maria Cândida R. Facciotti4 & Gabriel Padilla1,∗ 1 Department

of Microbiology, 2 Biotechnology Program, 3 Department of Immunology, Institute of Biomedical of Chemical Engineering, Polytechnic School, University of São Paulo, São Paulo, CEP Science; 05508-900, SP, Brazil ∗ Author for correspondence (Fax: +55 11 30917420; E-mail: [email protected]) 4 Department

Received 4 July 2002; Revisions requested 17 July 2002; Revisions received 30 August 2002; Accepted 2 September 2002

Key words: anthracycline, DNA intercalation, gel mobility shift, Streptomyces

Abstract An improved, rapid and inexpensive gel mobility shift assay was developed for the screening of anthracycline antibiotics. The assay based on the intercalation activity of these molecules into dsDNA was used to assess the activity of partially purified antibiotics. Detection limits were of 0.1 ng ml−1 with an average run time of 2 h. The assay is potentially useful for high throughput screening in bioprospecting, for monitoring fermentation production phases and downstream purification process.

Introduction The anthracycline antibiotics are effective antineoplasic drugs, widely used for the treatment of a variety of solid tumors and hematological malignancies. The first identified anthracycline molecules of chemotherapeutic use daunorubicin (daunomycin) and doxorubicin (adriamycin), were isolated from the pigment-producing bacterium Streptomyces peucetius in the early 1960s, and remain widely used today in clinical treatments (DiMarco et al. 1964, Arcamone et al. 1969a,b, Strohl et al. 1997). These molecules are composed of a tetracyclic chromophore group (aglycone) to which different glycosidic groups can be attached (Strohl et al. 1990), the latest being responsible for the recognition of the biological target (Mendez & Salas 2001). A number of studies have shown that the primary mode of action of the anthracyclines can be attributed to their intercalation into DNA. This intercalation inhibits macromolecular biosynthesis and stabilizes the topoisomerase II-DNA complex, which induces DNA strand breakage (Drlica & Franco 1988, Culliname et al. 1994, Pommier 1995, Wang et al. 1995, Horto-

bágyi 1997, Strohl et al. 1997, Gewirtz 1999, Wang et al. 2001). The affinity of certain anthracyclines molecules varies according to the target molecule. An example is the doxorubicin sequence specificity of DNA > cardiolipin > RNA (Mustonen & Kinnunen 1993, Doroshow 1995). The intercalation of the aglycone portion of the anthracycline, between adjacent DNA base pairs has as preferential sequence 5 (A/T)C-G and 5 (A/T)-G-C (Cutts & Phillips 1995, Chaires 1995, Rosmalen et al. 1995, Strohl et al. 1997). Molecules, such as daunorubicin, ditrirubicin B, and aclarubicin, that contain one, two, or three sugars, respectively, showed a binding preference for intercalation between 5 GT 3 (Shelton et al. 1996). The number of sugars contained in the molecules can protect bases in the DNA. Molecules with 1 sugar protect 3 bases, with 2 sugars protect 4 bases and with 3 sugars protect of 6–8 equal base pairs (Shelton et al. 1996). Toxicity and resistance of several cancer cell lines encourages the search for both more active and less toxic compounds (Strohl et al. 1997, Amarante-Mendes & Green 1999, Arola et al. 2000, Horenstein et al. 2000, Shadle et al. 2000).

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Fig. 1. Minimal intercalation concentration assay (MICA) of retamycin. (A) Ladder 1 kb, (B) pUC 18. Samples 1–10 with concentrations of 0.1 to 1 ng retamycin ml−1 .

Materials and methods DNA mobility shift assay

Fig. 2. Antibiotics shift mobility assay. (A) Ladder 1 kb, (B) pUC 18, (1) retamycin, (2) doxorubicin, (3) ciclamycin, (4) actinomycin D, (5) cycloheximide, (6) rifampicin. Concentration 10 ng ml−1 .

A frequent problem encountered during screening programs concern the use of simple and reliable methods to select new potential anthracyclines. Several methods have been used for screening of anthracyclines and other antitumoral drugs. In general, these methods are time-consuming, expensive, difficult to perform, and poorly reproducible, limiting their use for high-throughput assays (Mirabelli et al. 1985, Gibbs et al. 1994, Hotta et al. 1995, Haselsberger et al. 1996). In this work, we report the use of an improved and unexpensive gel mobility shift assay, that can be use for the rapid screening of the anthracyclines, based on the intercalation activity of these molecules into ds DNA. This assay can be applied to high throughput screening in bioprospecting, for monitoring fermentation production steps and downstream purification process.

BamHI linearized plasmids, pUC 18 (2,6 kb) and pET23 (3,66 kb) were used for dsDNA intercalation. Plasmid DNA isolation was carried out using a Flex Prep kit (Amersham-Pharmacia Biotec), following the manufacturer instructions. Assays were conducted using 1 µl (100 ng µl−1 ) plasmid DNA mixed with 5 µl extracted supernatants or intracellular samples. The final volume of each reaction was made to 10 µl with MilliQ water. The mixtures were incubated for 15 min at 37 ◦ C. Electrophoretic assays were run using a 1% (w/v) agarose gel without ethidium bromide and 0.5× TBE buffer at 5.3 V/cm for 2 h. To determine the minimal concentration of the antibiotic detected by the assay, purified retamycin samples ranging between 0.1 to 1 ng µl−1 were used following the above procedure. To assess the intercalation activity of different antibiotics, 10 ng µl−1 of purified reference samples of doxorubicin, retamycin and commercial antibiotics were assayed according to the standard protocol. Streptomyces strains Anthracycline producing strains: Streptomyces olindensis CCT (Tropical Culture Collection) 4859 (retamycin), S. olindensis ICB 20 (retamycin, mutant isolated from CCT 4859), S. violasceus CCT 5016 (rhodomycin), S. purpurascens CCT 5014 (rhodomycin A and B), S. capoamus CCT 5122 (cyclamycin), and S. peucetius ATCC 29050 (doxorubicin). No anthracycline producers strains: S. avermitilis ATCC 31267 (avermectin), S. argillaceus ATCC 12956 (mitramycin), S. olivaceus TU 2356 (elloramycin), and S. lividans TK64. Strains were grown in R5Mod (similar to R5 medium described by Kieser

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Fig. 3. Mobility shift of Streptomyces antibiotic producers. (A) DNA Ladder 1 kb; (B) control (pUC 18) supernatant and intracellular samples: (1, 9) S. olindensis 4859; (2, 10) S. olindensis ICB20; (3, 11) S. violasceus 5016; (4, 12) S. purpuracens 5014; (5, 13) S. argilaceus 12956; (6, 14) S. olivaceus 2356; (7, 15) S. avermitilis 31267; (8, 16) S. lividans TK 64.

et al. 2000; modified with Tris 3.09 g l−1 instead TES buffer and sucrose) for 5 d at 30 ◦ C and 200 rpm. Antibiotic samples Retamycin was purified by HPLC. Ciclamycin was a kind gift from the Antibiotics Department at Federal University of Pernambuco (Brazil). Doxorubicin was a gift from Zodiac Laboratories (Brazil). Actinomycin D, cyclohexamide and rifampicin were purchased from Sigma. Purification procedures for secreted and intracellular antibiotics Secreted and intracellular antibiotics, were extracted according to the following protocols: 1 ml of culture supernatant was treated with chloroform (v/v) to eliminate DNAses. Samples were mixed by vortexing until pigments were extracted into the aqueous phase. Chloroform extracts were concentrated by speed-vacuum, and suspended in 500 µl MilliQ water. Intracellular antibiotics were extracted by addition of 2 ml methanol per g wet cells. Methanolic extracts were concentrated and dissolved in 500 µl MilliQ water. To obtain aglycone molecules, samples were treated with HCl which hydrolyses sugars from the aglycone moiety. Fermentation assay in bioreactor Growth and retamycin production by S. olindensis ICB20 mutant were determined using a 15 l Biolafitte bioreactor with working volume of 9 l of R5 mod

medium; 1 ml of reactivated cells grew for 24 h, prior inoculation. Conditions were: 500 rpm agitation at 30 ◦ C for 48 h, aeration 1 vvm (volume of dissolved O2 /volume of medium per min), and pH 7. Samples were collected at intervals for 48 h and analyzed for dry mass, substrate consume (enzymatic method for GOD-POD Enzyme kit E-CELM), retamycin production by spectrophotometry (490 nm), intercalating activity was analyzed by gel-shift electrophoresis as above described. Apoptosis assay The experiments were performed on human promyelocytic HL60 cells. The cells were cultured in RPMI 1640 (Gibco), supplemented with 10% (v/v) fetal calf serum (Gibco), 1% (w/v) glutamine, penicillin 100 units ml−1 and streptomycin 100 µg ml−1 . Apoptosis effect was determined by a DNA fragmentation assay (Nicoli et al. 1991). Briefly, aliquots of 1.5 × 105 cells ml−1 plated in 96-well plates and cultured for 16 h with various concentrations of drugs were harvested and suspended in a hipotonic fluorescence solution HFS (0.1% sodium citrate, 0.1% Triton X-100 and 50 µg propidium iodide ml−1 ). Samples were analyzed on a FACScan flow cytometer (Bacton-Dickinson Mountain View, CA).

1810 (Figure 2). This technique can also be used for a screening program of potential producers of intercalating molecules, the method described here is very useful, because it allows the detection of low activities between the producer strains. A gel shift assay with products isolated from different Streptomyces strains (Figure 3), shows high intercalation with maximum mobility shift alteration of 1.5 kb for extracellular products of S. violascens 5016, S. purpurascens 5014, S. olindensis 4859, and S. olindensis ICB20. Intermediate alterations of 0.5 kb were observed with intracellular products of the same producers. No activity was detected for S. argillaceus 12956, S. olivaceus 2356, S. avermitilis 31267 and S. lividans TK24. Gel mobility shift assay for purification and downstream process

Fig. 4. Mobility shift of retamycin samples from S. olindensis ICB20 extracted with methanol and methanol/HCl (a). (A) DNA Ladder 1 kb; MeOH treatment (1) 10 ng µl−1 ; (2) 30 ng µl−1 ; (3) 50 ng µ−1 ; (4) 70 ng µl−1 ; (5) 100 ng µl−1 ; MeOH/HCl treatment (6) 10 ng µl−1 ; (7) 30 ng µl−1 ; (8) 50 ng µl−1 ; (9) 70 ng µl−1 ; (10) 100 ng µl−1 ; (B) pUC 18, and quantification of apoptosis in HL-60 cells incubated with various concentrations of retamycin extracted with meltanol (solid bars) or meltanol/HCl (open bars).

Results and discussion Detection levels of the gel mobility shift assay The sensitivity of the method is very high and was demonstrated through a minimal intercalation concentration assay (MICA), where retamycin samples with concentrations between 0.1 ng ml−1 until 1 ng ml−1 showed a shift of 1 kb (Figure 1). The intercalation property is a fundamental key to identify molecules with the potentiality of antitumoral activity. To demonstrate this hypothesis several antibiotics were studied and alterations of the mobility were only observed for recognized antitumoral compounds doxorubicin, retamycin and actinomycin D, while control antibiotics cyclohexamide and rifampicin were negative

Another application of this technique is related to screening of samples from purification or downstream processes. During these procedures, several organic solvents are used mainly: methanol, chloroform, acetone, HCl, and trifluoracetic acid for HPLC. Some of these solvents can degrade the antibiotic molecule, leading to loss of intercalation and biological activity. Figure 4a shows the results of retamycin purification with methanol and methanol/HCl as solvents. Samples 2–6 treated with methanol had a mobility alteration of 0.5 to 1 kb, whereas samples 7–11 treated with methanol/HCl did not show any DNA shift, probing that the latest treatment led to inactivation of the compound. To detect if samples still keep biological activity after solvent treatment, induction of apoptosis was carried out through measuring it in HL60 cells. Samples treated with methanol showed cell death induction from 100 ng ml−1 (40% cell death) until 400 ng ml−1 (98% cell death); while samples treated with methanol/HCl loose completely their biological activity as consequence of the hydrolysis (Figure 4b). DNA intercalation using the aglycone chromophore is a requirement for the DNA binding of anthracycline antibiotics, and most probably is needed for their biological activities, is not sufficient since the aglycone alone is not an active anticancer agent. The sugar moieties besides the intercalator chromophores play essential roles in deciding whether these compounds possess antitumor activity or not. Bioactivity of anthracyclines requires optimal fit of the sugar at the interface between DNA and topoisomerase (Wang et al. 1995, Zunino et al. 2001).

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Fig. 5. Production of retamycin by Streptomyces olindensis ICB 20 (a). () = Biomass (g l−1 ); () = consumption of carbon source (g l−1 ) and () = retamycin production (mg l−1 ), medium R5Mod, and mobility shift of supernatant samples from a bioreactor (b). (A) DNA Ladder 1 kb; (B), pET 23, samples time 0 to 48 h.

Gel shift assay for screening of anthracycline producer strains With the purpose of developing a rapid and inexpensive method to assess production of DNA intercalating molecules in fermentation process or screening programs, the DNA mobility assay showed to be very useful. To detect when a producer of intercalating antibiotics initiates the biosynthesis, samples from a bioreactor can be collected, purify partially and the production detect by spectrometry and/or gel mobility shift. In this work, a fermentation of the retamycin producer S. olindensis ICB20 was carried out. Samples were collected at different intervals of time. Initial production was detected by spectrophotometry after 28 h (Figure 5a). This result was confirmed by gel

mobility shift assay, where initial alterations in DNA migration were also observed at 28 h. The shift was more evident at 32 h with a very clear mobility of 1 kb when compared with control sample (Figure 5b). In addition noticeable alterations in the ethidium bromide staining were also observed. These alterations are related with the saturation of DNA binding sites by the anthracycline molecules reducing the number of available sites for ethidium bromide. DNA bands are fainter on assays with samples between 40–48 h compared to samples of 16 to 28 h. During the fermentation process, the cells synthesize many enzymes being some of them able of degrade DNA. Several procedures were tested in order to block DNase activity, such as sample treatment with EDTA, filtration, high

1812 temperature treatment and chloroform extraction; the latest showed the best results, due to its simplicity and rapid inactivation of Dnases. The gel mobility shift assay described is easy, rapid and low cost, when compared with other methods such as PCR inhibition (Hotta et al. 1995), which requires many reagents and takes longer time due to the sample purification and amplification cycles. High temperatures (e.g., PCR denaturing cycle at 95 ◦ C) may also affect antibiotic stability and binding properties. In conclusion, our technique is potentially useful in studies of intercalating molecules for high throughput screening in bioprospecting, for monitoring fermentation production phases and downstream purification process.

Acknowledgements Authors want to thank Drs Inês Chen and Gilson P. Manfio for suggestions and manuscript discussions, Drs Roberto Berlinck, Alfredo Braña and José Salas for the antibiotic purification, Mrs Sileine Costa Rodrigues and Juliana Ferreira de Melo for technical assistance, and FAPESP for financial support to RLAF (97/14412-4) and LMG (00/07288-0).

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