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Immunocytochemistry for Drugs Containing an Aliphatic Primary Amino Group in the Molecule, Anticancer Antibiotic Daunomycin as a Model

Kunio Fujiwara, Hironori Takatsu and Kazuhiro Tsukamoto

Department of Applied Life Science, Faculty of Engineering, Sojo University, Kumamoto, Japan (KF); and Department of Pharmacotherapeutics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan (HT,KT)

Correspondence to: Kunio Fujiwara, Department of Applied Life Science, Faculty of Engineering, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan. E-mail: fujiwara{at}life.sojo-u.ac.jp

	Summary

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An immunocytochemistry (ICC) for the anticancer antibiotic daunomycin (DM) was developed using a combination of anti-DM serum produced against N-(gamma-maleimidobutyryloxy)succinimide (GMBS)-conjugated DM, and DM-uptake human melanoma BD cells. The antiserum was demonstrated to be specific for DM and the structurally related analogs adriamicin and epirubicin by an ICC model system of the enzyme immunoassay (EIA) using glutaraldehyde (GA)-conjugated DM as a solid phase antigen. No cross-reaction occurred with any of the other antibiotics tested such as bleomycin, pepleomycin, and mitomycin C. Successful DM ICC required a series of processes prior to the immunocytochemical reaction: the cells were first fixed with GA, then reduced with NaBH₄, treated with hydrochloric acid, and finally digested with protease. The cell specimens were then subjected to immunoreaction with anti-DM serum followed by peroxidase-labeled goat anti-rabbit IgG/Fab', and in both immune reagents the detergent Triton X-100 was contained as well. The present ICC covering all these processes successfully stained for DM in the nucleus and in the perinuclear Golgi region of the cytoplasm of the BD cells, consistent with the results obtained by the DM autofluorescence method. This ICC was found to be three times as sensitive as the cytofluorometric method and applicable to the paraffin sections of the liver of rats 24 hr after an IV injection of DM. The principle used in the present study for developing DM ICC might be applied to other drugs containing the primary amino group(s) in the molecule. Thus, these ICCs for drugs are direct, precise and easy new methods that should have potential for pharmacology and toxicology studies of drugs, revealing the localization of a drug in cells and tissues. (J Histochem Cytochem 53:467–474, 2005)

Key Words: daunomycin • immunocytochemistry • subcellular localization

	Introduction

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THE USEFULNESS of immunocytochemical (ICC) studies for small-sized molecules of the biogenic endogenous amines or amino acids such as serotonin, histamine, epinephrine, polyamines, GABA, and glutamic acid is evident from the many reports published on this subject (Decavel et al. 1987; Nilsson et al. 1987; Popratiloff et al. 1996; Fujiwara et al. 1998). Success in these studies suggests a possibility for developing ICC for drugs in the same way, revealing their topographical distribution in cells and tissues. To our knowledge, however, there are only a few reports concerning ICC for drugs such as methamphetamine (Ishiyama et al. 1987), gentamycin, tobramycin, and vancomycin (Beauchamp et al. 1991,1992). In these reports, detailed fundamental conditions necessary for ICC, such as a variety of treatments of cell and tissue specimens with some reagents prior to or during immunoreaction, have not been fully described. Autoradiography methods, which are commonly employed for pharmacological studies on drugs, are sensitive and specific but possess drawbacks inherent in the use of a radioisotope. They also require time-consuming and complicated techniques and provide only an indirect detection system for drug localization in cells and tissues (Peng and Nimni 1999; Mountfield et al. 2000). Autofluorescence is also used as a marker in the case of some drugs, but it is lacking in sensitivity (Egorin et al. 1974,1980; Krishan et al. 1976; Paschoud et al. 1985; Willingham et al. 1986; Rutherford and Willingham 1993).

To overcome each of these drawbacks and to develop an easy and generally applicable method revealing a precise localization or uptake of the drug in the cells and tissues, we established the present ICC method. In this study we used an anthracycline antibiotic daunomycin (DM) as a prototype, which has the characteristic of possessing an aliphatic primary amino group in the molecule and the autofluorescence as well. This first characteristic may permit the drug to be covalently fixed in situ with a fixative, and the second characteristic may be used to compare the presently developed ICC methods with the cytofluorescence method.

	Materials and Methods

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Chemicals
Daunomycin HCl, epirubicin HCl and adriamycin HCl were generously supplied by Meiji Seika Co. Ltd., Tokyo, Japan; Pharmacia Co. Ltd., Tokyo, Japan; and Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan, respectively. Glutaraldehyde (GA, 25% in water), formalin (37%) and Triton X-100 were obtained from Nacalai Tesque (Kyoto, Japan). Sodium borohydride (NaBH₄) and protease (Type XXIV: Bacterial) were from Sigma-Aldrich (St Louis, MO).

Antibody
Anti-DM serum was produced in a rabbit against DM conjugated to bovine serum albumin (BSA) using a heterobifunctional agent N-(gamma-maleimidobutyryloxy)succinimide (GMBS), as shown in our previous paper (Fujiwara et al. 1981).

Synthesis of Daunomycin-glutaraldehyde-bovine Serum Albumin Conjugate (DM-GA-BSA)
BSA (10 mg) in 1.0 ml of 1 M sodium acetate and 1.0 ml of 3 mM GA were mixed and incubated at room temperature for 30 sec by stirring. DM (1 mg) in 1.0 ml of 1 M sodium acetate, pH 8.5, was then added to this mixture. This was followed by incubation for 30 min before 1 M monoethanol amine (1 ml) was added to terminate the reaction. The reaction mixture was further incubated for 30 min and was dialyzed against 10 mM Tris-HCl buffer, pH 7.2, for 6 hr at 4C.

EIA method
The wells in microtiter plates (Nunc F Immunoplates I; Nunc, Roskilde, Denmark) were coated by loading 100 µl of daunomycin-glutaraldehyde-bovine serum albumin (DM-GA-BSA) conjugate (10 µg/ml) in 10 mM Tris-HCl buffer, pH 8.5, containing 10 mM NaCl and 10 mM NaN₃ and being left for 20 min at 37C. After they were rinsed with 10 mM phosphate buffer, pH 7.4, the plates were incubated with 100 µl of 60 mM phosphate buffer, pH 7.4, containing 1.0% casein and 0.1% NaN₃ for 1 hr at 37C to avoid nonspecific adsorption of serum components in the samples. The wells were then incubated overnight at 4C with 100 µl of anti-DM serum, diluted to various degrees with 20 mM phosphate buffer, pH 7.0, containing 0.15 M NaCl, 0.05% Tween 20, and 0.02% BSA, followed by goat anti-rabbit IgG labeled with horseradish peroxidase (HRP, 1:2000) for 1 hr at 25C. The amount of enzyme conjugate bound to each well was measured with o-phenylenediamine as a substrate, and the absorbance at 492 nm was read with an automatic ELISA analyzer.

EIA Inhibition Test
Wells in a microtiter plate were coated with 100 µl of the DM-GA-BSA conjugate (10 µg/ml) as described above. The wells were then incubated with 50 µl of a fixed concentration of anti-DM serum (1:5000) and 50 µl of different compounds (DM, adriamycin, epirubicin, bleomycin, pepleomycin, mitomycin C, streptomycin, or gentamycin) at various concentrations overnight at 4C, followed by incubation with goat anti-rabbit IgG (1:2000) for 1 hr at 25C. The bound HRP activity was measured as described above.

Cells
Human melanoma BD cells were grown on 18 x 18-mm coverslips in MEM medium with 10% calf serum (BioWhittaker; Walkersville, MD), penicillin (50 U/ml), and streptomycin (50 µg/ml) in a 95% air/5% CO₂ atmosphere, and used 24 hr after plating. DM in aqueous stock solution at 1 mg/ml was then added to the cells to a final concentration (0.03 to 3.0 µg/ml) and further incubated for 2 hr at 37C.

Immunocytochemistry
The DM-uptake BD cells were washed in Mg²⁺- and Ca²⁺-free PBS and then immersion fixed in 1.0% to 3.0% GA in 0.1 M sodium phosphate buffer, pH 7.6, at 25C for 30 min. The specimens were treated with a series of 0.05% NaBH₄ in TBS (50 mM Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl) for 5 to 20 min, 0.5–4 N HCl for 30–90 min, and 0.001–0.004% protease in TBS for 30 min. During each process of the treatment the specimens were washed three times with TBS. Next, the specimens were blocked with a protein solution containing 10% normal goat serum, 1.0% BSA, and 0.1% saponin in TBS for 1 hr at room temperature and then directly incubated at 4C overnight with anti-DM serum (diluted 1:2,000–10,000 with the above protein solution supplemented with 0.1% Triton X-100). Specimens were washed with TBST (TBS containing 1% Triton X-100) three times, 5 min at a time, and then incubated with a horseradish peroxidase-labeled goat anti-rabbit-IgG (whole IgG; Cappel, West Chester, PA) 1:500 or IgG/Fab' (MBL; Nagoya, Japan) 1:300 for 1 to 24 hr at 4C. After rinsing with TBS, the site of the antigen–antibody reaction was revealed for 10 min with diaminobenzidine and H₂O₂ (Fujiwara and Masuyama 1995).

Tissue Materials
Male Wistar rats (250 g body weight) were injected IV with DM at a dose of 4 mg in 0.8 ml of PBS. Twenty-four hr after the injection, the rats were anesthetized with pentobarbital intraperitoneally, perfused transcardially with 100 ml of saline for 2 min, and subsequently fixed by perfusion with 300 ml of 2% GA in 0.1 M phosphate buffer, pH 7.4, for 4 min at room temperature. Specimens of the liver were postfixed in the same fixative overnight at 4C and subsequently routinely embedded in paraffin. Five-µm paraffin sections were deparaffinized and then processed for DM ICC as described above.

Control Experiments
In the DM immunocytochemistry study, the specificity of immunostaining was ascertained by incubating control specimens with the secondary antiserum alone, the anti-mitomycin C serum (Fujiwara et al. 1982), and DM serum preabsorbed with DM-GMBS-BSA conjugate at a concentration of 10 µg/ml.

Fluorescence Microscopy
This was performed essentially according to the method of Rutherford and Willingham (1993). The DM-uptake human melanoma cell line sk-mel-37 cell specimens were either fixed with 3.7% formaldehyde in PBS for 10 min or GA (1–2%) in PBS for 30 min at room temperature, washed with PBS, and examined with an Olympus epi-fluorescence microscope equipped with a 100-W high-pressure mercury lamp light source and filters for excitation wavelength of 490 nm and for emitted light >515 nm.

	Results

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Antibody Dilution
Anti-DM serum produced against GMBS-conjugated DM (DM-GMBS-BSA conjugate) was characterized by an EIA system employing GA-conjugated DM (DM-GA-BSA conjugate) as the solid phase antigen. This EIA system may be useful as a model of DM ICC, because the ICC is based on the principle that DM in situ is coupled to the tissue proteins with the fixative GA through covalent bonds. As shown in Figure 1, significant binding activity was observed at serial dilutions of anti-DM serum, even at more than 10,000 times dilution. On the other hand, a much lower level of immunoactivity was seen in an EIA system using the solid phase antigen of BSA itself at the same concentration (10 µg/ml) (Figure 1), this showing binding activity of the antibody produced against the carrier BSA molecule of DM-GMBS-BSA conjugate used as the DM antigen. Thus, DM immunoreactivity was indicated as the remainder of their binding activity.

View larger version (16K): [in this window] [in a new window]   Figure 1 Dilution curves for anti-DM serum: several dilutions of anti-DM were incubated in microtiter wells coated with either DM-GA-BSA conjugate or BSA at a concentration of 10 µg/ml each overnight at 4C, followed by the procedure described for the EIA using HRP-labeled goat anti-rabbit IgG (whole) (diluted 1:2000) as the second antibody. Closed circle, DM-GA-BSA conjugate; open circle, BSA.

View larger version (17K): [in this window] [in a new window]   Figure 2 Reactivity of anti-DM serum as measured by its immunoreactivity in the EIA inhibition test. The curves show the amount (percentage) of bound enzyme activity (B) for various doses of DM (closed circles), adriamycin (open circles), or epirubicin (closed triangles) as a ratio of that bound using the HRP-labeled second antibody alone (Bo).

The best experimental conditions for DM ICC were reached by determining an optimal concentration of sodium borohydride, HCl, and protease, and an optimal incubation time for their reaction with the cells. After several sets of ICC conditions were tried, it was found that a 5-min incubation with a 0.05% NaBH₄ solution, a 30 min-incubation with 1 N HCl solution, and a 30 min incubation with a 0.004% protease solution provided an effective ICC system (Table 1). Also, the present indirect immunoperoxidase procedure for DM ICC needed Triton X-100 at a concentration of 0.1% in an antibody solution for both the first and second immune reaction (Table 1). Furthermore, it was revealed that horseradish peroxidase-labeled goat anti-rabbit IgG/Fab' was preferable to the whole IgG as the second antibody in the ICC (Table 1). On the other hand, when the pretreatment of either HCl or protease was omitted from the ICC protocol, DM immunostaining, especially in the nuclei, was very weak or non-existent (Table 1 and Figure 3b). Also, the elimination of borohydride reduction resulted in nonspecific staining of the cells, and to contrast, the reduction with 0.05% NaBH₄ longer than 5 min resulted in weaker immunostaining.

View larger version (55K): [in this window] [in a new window]   Figure 3 Immunostaining and fluorescence of DM in the DM-uptake human melanoma BD cells. DM immunoreactivity was seen both in the nuclei and paranuclear Golgi regions under the presently established ICC conditions (a), but only in the Golgi regions when the hydrochloric acid treatment step was omitted from the ICC protocol (b). The staining (seen in a) was completely abolished by absorption of the anti-DM serum with DM-BSA conjugate prepared via GMBS (DM-GMBS-BSA conjugate, 10 µg/ml) (c). Fluorescence of DM was seen only in the nuclei of the cells fixed with 3.7% formaldehyde (d), and only in the paranuclear Golgi regions of the cells fixed with glutaraldehyde (e). Arrows, nuclei; arrowheads, Golgi regions. Bar = 50 µm.

To compare the results of ICC, the cytofluorometric method was also employed using the melanoma cells pretreated with DM as above. In the cells fixed with 3.7% formaldehyde, the orange-colored fluorescence of a DM signal was clearly seen only in the nuclei of the cells (Figure 3d). Furthermore, in the cells fixed with 1% GA, strong autofluorescence of the green signal, most of which may be due to the autofluorescent mitochondria (Rutherford and Willingham 1993), occurred in the cells, making it difficult to distinguish it from a DM signal: the DM signal was very slight or nonexistent in the nuclei, though it was significant in the Golgi regions of the cells (Figure 3e). However, the green autofluorescence disappeared very rapidly after irradiation by excitation UV light, while some parts of the DM signal were also affected (Figure 3e).

Specificity and Sensitivity of ICC
Anti-DM serum specifically stained for DM analog adriamycin or epirubicin taken up in the melanoma cells under the same conditions as for DM, the immunostaining pattern of each being completely the same as with DM (data not shown). The DM ICC was sensitive enough to stain for DM in the melanoma cells whose treatment with DM was as little as 30 ng/ml for 2 hr by the imidazole–3,3'-diaminobenzidine tetrahydrochloride procedure. No cross-reactivity was seen with any of the compounds tested (bleomycin, mitomycin C, or streptomycin). Using the cytofluorometric method for the cells fixed with 3.7% formaldehyde, DM could be detected in the cells that had been treated with as little as 100 ng of DM/ml for 2 hr. In the cells fixed with GA, on the other hand, no DM signal was observed in the cells pretreated even to the degree of 300 ng of DM/ml for 2 hr.

Evaluation of Tissue Staining
The optimal conditions for DM ICC established above were applied to ICC for the paraffin sections of the liver of rats 24 hr after an IV injection of DM. Strong immunoreaction for DM was observed in Kupffer cells, bile duct cells, smooth muscle cells surrounding the branch of the hepatic artery, and certain cells in the connective tissues of Glisson's capsule. Less strong staining was seen in the endothelial cells in the branches of the portal vein and hepatic artery (Figure 4a). Only very slight staining was found in the nuclei of the hepatic cells but almost not at all in the cytoplasm (Figure 4a). Conventional immunocytochemical staining controls (second level controls and absorption controls) were all negative (Figure 4b).

View larger version (164K): [in this window] [in a new window]   Figure 4 Rat liver stained for DM by immunocytochemistry (indirect peroxidase method). The staining (a) was completely abolished by absorption of the serum with 10 µg/ml of DM-GMBS-BSA conjugate (b). Arrows: 1, Kupper cells; 2, nuclei of hepatic cells; 3, endothelial cells of portal vein; 4, bile duct cells; 5, connective tissue cells; 6, smooth muscle cells. Bar = 50 µm.

	Discussion

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

The detailed conditions for establishing immunocytochemistry for drugs have not yet been elucidated. The anti-DM serum used in the present ICC study was a sample of an antibody produced against GMBS-conjugated DM and, therefore, was tested for binding activity by the EIAs with GA-conjugated DM as the solid phase antigen (Figure 1 and Figure 2). These EIA tests could be useful if they indicate whether or not the antibody reacts with DM in cells and tissues used for the present ICC study using GA as a fixative. The anti-DM serum showed a much higher titered binding activity to DM-GA-BSA conjugate than BSA itself at the same concentration (Figure 1). Furthermore, it was demonstrated by the EIA inhibition test that the antibody binding was inhibited by DM to the highest degree, followed by the structurally related analogs adriamycin and epirubicin with a cross-reactivity of 71% and 62%, respectively (Figure 2). Also, no inhibition was evident with the other antibiotics, mitomycin C, bleomycin, pepleomycin, and actinomycin D (data not shown).

In ICC studies for small-sized drug molecules, a drug that has been specifically distributed according to its characteristic into the cells should be fixed in situ without redistribution during fixation. Thus, the fixation must be rapid and effective. Immersion fixation of the culture cells with 1–2% GA for 30 min fulfilled these requirements, as the immunocytochemical staining revealed a well-localized and specific reaction for DM in the nucleus and perinuclear Golgi region in the cytoplasm of the melanoma BD cells (Figure 3a). However, weaker fixatives often resulted in signs of diffusion artifacts such as weak nuclear staining and diffuse cytoplasmic staining (data not shown). The present immunostaining pattern agrees well with the results of the DM autofluorescence method. Specifically, the DM signal localized only in the nucleus of the cells when fixed with 3.7% formaldehyde, whereas it did so mainly in the perinuclear Golgi region (Figures 3d and 3e) with GA fixation, these being completely consistent with the results that Rutherford and Willingham (1993) reported for DM localization in the culture cells. In other ICC studies, GA has proved useful as a fixative for small molecular compounds with the amino group(s) such as the biogenic endogenous amines (Decavel et al. 1987; Nilsson et al. 1987; Fujiwara et al. 1996,1998) and amino acids (Hepler et al. 1988; Popratiloff et al. 1996; Spirou and Berrebi, 1997).

Hydrochloric acid treatment (1 N for 30 min) and protease digestion (Bacillus amyloliquefaciens, 0.004% for 30 min) for the cell specimens prior to the immunocytochemical reactions, together with an addition of a detergent (0.1% Triton X-100) to the immune reagents greatly improved the uniformity and intensity of the staining (Table 1). All these treatments of cell specimens might enhance the permeability of the antibody into the cells. In previously reported ICC for bromodeoxyuridine, pretreatment of paraffin sections with protease and HCl in combination proved useful for the restoration of the antigenicity in the nuclei of the cells, possibly due to the denaturation of the DNA that is necessary for the antibody to react with bromodeoxyuridine (Moran et al. 1985; Sugihara et al. 1986; Hayashi et al. 1988; Hume and Keat 1990). Similarly, in the present ICC, DM immunoreactivity in the nuclei of the cells was hardly detected when the hydrochloric acid treatment or protease digestion was omitted from the present ICC protocol, although the perinuclear Golgi region was strongly stained (Table 1 and Figure 3b). Furthermore, it was found that the second immunocytochemical reaction occurred more easily with the anti-rabbit IgG/Fab' than with the whole IgG, possibly due to the smaller molecular size of the fragmented antibody. Sodium borohydride has been used extensively to decrease the nonspecific immunobinding in ICC using GA as a fixative, as the borohydride reduces a chemically active aldehyde group of GA that remains in excess in cells after fixation (Willingham 1983; Fujiwara and Masuyama 1995). In the present study, however, NaBH₄ might have affected the antigenicity of DM in situ, as it also reduces the anthracycline ring moiety of a DM molecule. Hence, it was determined that the concentration of NaBH₄ (0.05%) and its 5-min incubation provided the optimal reduction system that hardly affected the DM antigenicity but degraded almost all the active aldehyde group of GA.

Under the optimal conditions established for DM ICC, preliminary experiments were examined using paraffin sections of the liver of rats 24 hr after a single IV injection of DM. It was found that anti-DM serum immunostained for DM in Kupffer cells, the endothelial cells of blood vessels as well as their neighboring cells (smooth muscle cells, bile duct cells, and connective tissue cells) in the Glisson's capsule. This may suggest that Kupffer cells actively endocytize DM injected into the bloodstream. We are now undertaking toxicology studies on DM especially for its specific localization in the kidney and heart, which may help develop a better understanding of the mechanism for the unique renal and cardiac toxicity of DM (Tan et al. 1967; Bachur 1973; Lefrak et al. 1973; Steinherz et al. 1991) (Fujiwara K et al., unpublished data).

In conclusion, DM ICC method was newly developed with an advantage of being able to detect DM in the cells with a higher degree of sensitivity than the previously available cytofluorometric method. In addition, this ICC was demonstrated to be useful for the DM analogs adriamycin and epirubicin. The principle used in the present study for developing a DM ICC method might be applied to other drugs, especially for those containing primary amino group(s) in their molecules. Thus, these ICCs for drugs are direct, precise, and easy new methods that should have potential for pharmacology and toxicology studies of drugs, revealing the localization of the drug in cells and tissues.

	Acknowledgments

 
This study was supported in part by a grant from the Japan Society for the Promotion of Science (15590148).

We are grateful to Dr Masashi Shin of Sojo University for valuable suggestions throughout this study.

	Footnotes

 
Received for publication June 25, 2004; accepted November 10, 2004

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