In Vivo Mechanical Loading Modulates Insulin-Like Growth Factor Binding Protein-2 Gene Expression in Rat Osteocytes

Mechanical stimulation is essential for maintaining skeletal integrity. Mechanosensitive osteocytes are important during the osteogenic response. The growth hormone-insulin-like growth factor (GH-IGF) axis plays a key role during regulation of bone formation and remodeling. Insulin-like growth factor binding proteins (IGFBPs) are able to modulate IGF activity. The aim of this study was to characterize the role of IGFBP-2 in the translation of mechanical stimuli into bone formation locally in rat tibiae. Female Wistar rats were assigned to three groups (n = 5): load, sham, and control. The four-point bending model was used to induce a single period of mechanical loading on the tibial shaft. The effect on IGFBP-2 mRNA expression 6 hours after stimulation was determined with nonradioactive in situ hybridization on decalcified tibial sections. Endogenous IGFBP-2 mRNA was expressed in trabecular and cortical osteoblasts, some trabecular and subendocortical osteocytes, intracortical endothelial cells of blood vessels, and periosteum. Megakaryocytes, macrophages, and myeloid cells also expressed IGFBP-2 mRNA. Loading and sham loading did not affect IGFBP-2 mRNA expression in osteoblasts, bone marrow cells, and chondrocytes. An increase of IGFBP-2 mRNA-positive osteocytes was shown in loaded (1.68-fold) and sham-loaded (1.35-fold) endocortical tibial shaft. In conclusion, 6 hours after a single loading session, the number of IGFBP-2 mRNA-expressing osteocytes at the endosteal side of the shaft and inner lamellae was increased in squeezed and bended tibiae. Mechanical stimulation modulates IGFBP-2 mRNA expression in endocortical osteocytes. We suggest that IGFBP-2 plays a role in the lamellar bone formation process.
Mechanical stimulation is important for the maintenance of the skeleton of humans and other animals [1–9]. Bone serves as a structural support for the body and has the ability to modify its architecture throughout life. Mechanical stimulation regulates bone mass and architecture according to Frost’s mechanostat hypothesis. Physical activity results in increased bone formation and decreased bone resorption, whereas inactivity has the opposite effect [10, 11].
The growth hormone-insulin-like growth factor (GH-IGF) axis plays an important role in the regulation of bone remodeling. Of the insulin-like growth factors (IGFs), IGF-I is predominantly expressed in rat bone and IGF-II is predominantly expressed in human bone [12–14]. IGFs are involved in bone formation after mechanical stimulation. Mechanical loading upregulates IGF-I mRNA expression in osteocytes, as shown in rat vertebra by Lean and colleagues using the caudal vertebra compression model [15]. Northern blot analysis also showed enhanced periosteal IGF-I mRNA expression after mechanical stimulation [16]. Besides an increase in IGF-I mRNA synthesis, Bravenboer and colleagues reported upregulation of IGF-I protein concentration in bone after mechanical stimulation using additional weight bearing in running rats (rat-with-backpack) [17].
As in other tissues, the autocrine and paracrine actions of IGFs in bone are modulated by a family of six structurally related binding proteins, the IGF binding proteins (IGFBPs). IGFBPs can be synthesized by osteoblasts [18, 19] and are capable of modulating the effects of IGFs by potentiating or inhibiting their action; some IGFBPs also have IGF-independent actions that can either inhibit or stimulate cellular function.
Since rat osteoblasts do not express IGFBP-1 [18, 20, 21], it is most likely that IGFBP-1 does not play a significant role during bone formation after mechanical stimulation in rats. Although IGFBP-2 is the major IGFBP synthesized by rat osteoblastic cells [18], a role for IGFBP-2 during mechanical loading has not been established yet. IGFBP-3 has both inhibitory and stimulatory effects on bone cells. IGFBP-4, which is locally produced by osteoblasts, has an inhibitory effect on bone metabolism, whereas locally synthesized IGFBP-5 by osteoblasts has a stimulatory effect [18, 22, 23]. However, the effects of IGFBP-4 on osteoblasts are IGF-I-dependent, whereas IGFBP-5 probably acts as a store for IGFs in bone tissue [18, 22, 23]. IGFBP-6 is expressed in rat osteoblasts [24]. In conclusion, IGFBP-2 is the most abundant IGFBP in rat osteoblasts; however, the role of IGFBP-2 during bone formation after mechanical stimulation in vivo has not been determined yet. We hypothesized that IGFBP-2 mRNA expression level might be changed in bone cells during bone formation after mechanical stimulation.
The aim of this study, therefore, was to determine the localization of IGFBP-2 mRNA in the cortical tibial shaft after mechanical stimulation. To this end, we induced a single period of mechanical loading using the four-point bending model of Forwood and Turner [1, 2, 7]. This resulted in bone formation in the rat tibia 5–8 days after stimulation [25]. We developed an in situ hybridization method especially for bone tissue to detect the local osteogenic response at the cellular level 6 hours after a single period of dynamic loading.
Materials and Methods
Animals
Fifteen female 12-week-old Wistar rats (235 ± 12 g; Harlan, Zeist, The Netherlands) were randomly assigned to three weight-matched groups (n = 5/group): load, sham, and control. The animal experiment was in accordance with the governmental guidelines for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee of the VU University Medical Center (Amsterdam, The Netherlands).
In vivo Mechanical Loading
The right tibiae underwent “mediolateral” loading (load), sham loading (sham, in which the opposed pads were placed at the inner position, 11 mm apart), or no loading (control) using the four-point bending system of Forwood and Turner [1, 7]. Since loading will result in bending and squeezing of the tibia and sham loading only in squeezing of the tibia, the sham group was used as a control for the load group. The left tibiae served as contralateral controls. The four-point bending model [25] was used to generate a single period of dynamic loading of the right tibia in rats in vivo in order to detect acute changes of IGFBP-2 mRNA locally in bone tissue after stimulation by mechanical stress. The rats were subjected to a single episode of loading comprising 300 cycles (2 Hz) using a peak magnitude of 60 N. Forwood and Turner showed that a single loading session resulted in bone formation in the rat tibia 5–8 days after stimulation [25]. Characterization of the in vivo strain using the four-point bending system was reported by Akhter and colleagues [26]. Using the four-point bending system at our laboratory, we demonstrated bone formation at the endosteal surface of the rat tibia 15 days after a single loading session with a frequency of 2 Hz during 300 cycles and an applied peak magnitude of 60 N [27]. The loading experiment was performed under general anesthesia (2% isoflurane in 1 L/min O2 and 2 L/min N2O). The rats were killed exactly 6 hours after loading. This time point was based on the literature [15], which was confirmed by a time-course pilot experiment at our laboratory using real-time reverse-transcription polymerase chain reaction analysis (H. W. van Essen, personal communication) showing the highest IGF-I mRNA expression 6 hours after loading. Since the actions of IGFs are influenced by their IGFBPs, we examined IGFBP-2 mRNA expression at 6 hours after loading. The tibiae were dissected and immediately fixed in 4% (w/v) paraformaldehyde (buffered in phosphate-buffered saline [PBS], pH 7.4) at 4°C for 24 hours.
Tissue
After fixation, the tibiae were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) with 0.5% paraformaldehyde in PBS at 4°C for 4.5 weeks. Finally, the tibiae were washed in PBS and dehydrated through a series of ethanol and xylene at room temperature and embedded in paraffin.
As a positive control, brain tissue was used [28]. Brains were dissected rostrally to the cerebellum (interaural coordinate 0 mm) and the hippocampus (interaural coordinate 4 mm) in three coronal blocks and immediately fixed in 4% (w/v) paraformaldehyde (buffered in PBS, pH 7.4) at 4°C for 24 hours, followed by washing in PBS, dehydration through a series of ethanol and xylene at room temperature, and embedding in paraffin.
Reagents
All restriction enzymes and modifying enzymes were purchased from Roche Molecular Biochemicals (Mannheim, Germany), as well as digoxigenin-uridine triphosphate (UTP), antidigoxigenin Fab fragments, nitroblue tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and blocking reagent. Nylon membranes were purchased from Qiagen (Hilden, Germany). Polyvinyl alcohol was obtained from Aldrich (Milwaukee, WI). Euparal mounting medium was purchased from Chroma Gesellschaft (Waldeck, Division Chroma, Münster, Germany). Silane-coated glass slides were obtained from Sigma-Aldrich (St. Louis, MO).
Mouse IGFBP-2 cDNA was kindly provided by Dr. S. L. S. Drop and Dr. J. W. van Neck (Department of Pediatrics, Sophia Children’s Hospital, Erasmus University, Rotterdam, The Netherlands) via Dr. S. C. van Buul-Offers (Department of Metabolic and Endocrine Diseases, University Medical Center, Utrecht, The Netherlands).
Synthesis of Digoxigenin-Labeled Complementary RNA (cRNA) Probes
Standard in vitro transcription reactions were carried out using T7- and Sp6-RNA polymerase with digoxigenin-UTP as a substrate [29]. cDNA encoding mouse IGFBP-2 cDNA, corresponding to amino acid position 98–258 and showing 95% homology with the corresponding rat IGFBP-2 cDNA fragment, was used as a template for the synthesis of antisense and sense digoxigenin-labeled RNA probe. The probe was specific for the mRNAs analyzed. The IGBP-2 probe did not show cross-reactivity with different types of mouse tissues (spleen, thymus, and brain) [28, 30, 31].
Nonradioactive In Situ Hybridization
Serial, longitudinal tibial sections (5 μm), which were cut in the posterior-anterior direction, and cross-sectional control brain sections (5 μm) were mounted onto RNase-free silane-coated glass slides and dried at 56°C for at least 3 days. Corresponding sections of the right and left tibia of one rat were mounted on the same glass slide. In situ hybridization was performed on every fiftieth section with a total of five slides per tibia (i.e., sections I, II, II, IV, and V). All sections were dewaxed, rehydrated, and rinsed in water. The sections were pretreated with 0.2 N HCl for 15 minutes at room temperature, permeabilized in proteinase K (15 μg/mL) for 30 minutes at 37°C, and subjected to an acetylation treatment [32]. Sections were rinsed in 2× SSC (0.3 M sodium chloride and 0.03 M sodium citrate) and kept in this solution until the start of hybridization.
Hybridization was performed in a solution containing 50% formamide, 2× SSC, 1× Denhardt’s solution, 250 μg/mL transfer RNA, 480 μg/mL herring sperm RNA, 10% dextran-sulfate, and the mouse IGFBP-2 digoxigenin-labeled cRNA probe at concentrations of 500 pg/μL (brain) and 1,000 pg/μL (tibia). Sections were hybridized overnight at 53°C. After hybridization, sections were washed with 50% formamide in 2× SSC at the hybridization temperature for 30 minutes and treated with RNase A (1 unit/mL) for 30 minutes at 37°C. Subsequently, sections were rinsed in 2× SSC, treated with 1% blocking reagent for 30 minutes, and incubated with sheep antidigoxigenin Fab fragments conjugated with alkaline phosphatase (1:1,500) overnight at 4°C.
Chromogenesis was performed in the dark with 0.38 mg/mL NBT and 0.19 mg/mL BCIP in the presence of 6% (w/v) polyvinyl alcohol [33], resulting in a blue precipitate. Finally, sections were counterstained with nuclear fast red, dehydrated through a series of ethanol, and mounted with Euparal. Sense probes were used to investigate the level of nonspecific binding.
Quantification and Statistics
First, a semiquantitative screening of all animals per group was performed on the antisense and sense slides to collect the in situ hybridization data. Sections I-V of all tibiae were scored in a semiquantitative manner, which was defined, on a Nikon Eclipse E800 microscope (Uvikon, Bunnik, The Netherlands), as follows: no expression (0), low expression (1), average expression (2), and high expression (3).
Effects of mechanical loading were expected in the shaft of the tibiae. Therefore, a second quantitative evaluation of the endocortical osteocytes expressing IGFBP-2 mRNA was performed with Lucia G Version 4.82 (Uvikon) using coded slides of sections II and III. The defined area of interest, which comprised the loading zone, started below the primary spongiosa at the endosteal side of the shaft following the total length of the shaft to the distal side (total length maximal 3,852 μm). At the endosteal side of the shaft, all osteocytes, which were positioned within 100 μm of the endosteal surface, were included in the area of interest. Within the area of interest, total osteocyte number and total IGFBP-2 mRNA-positive osteocyte number were measured.
The paired-samples Student’s t-test and independent samples Student’s t-test were used for statistical analysis using SPSS (Chicago, IL) version 9.0 for Windows. P < 0.05 was considered to reflect statistical significance.
Photography
Brightfield photographs with different magnifications (objectives ×10, ×20, and ×40) were made using a Leica microscope (DM4000B) with a digital camera (Leica DC500) and Leica software IM50 (Leica Microsystems, Rijswijk, The Netherlands).
Results
Endogenous IGFBP-2 mRNA Expression in Tissue
The control brain showed IGFBP-2 mRNA expression in the choroid plexus (Fig. 1A) and the neurons of the medulla oblongata (data not shown). In the internal control, i.e., the growth plate, IGFBP-2 mRNA was located in chondrocytes of the proliferative and hypertrophic zone (Fig. 1C). In control tibiae, IGFBP-2 mRNA was expressed in osteoblasts, which were situated against the surface of trabecular bone (Fig. 1C, E) and the endosteal side of the shaft (Fig. 1G). IGFBP-2 mRNA expression was also observed in some trabecular osteocytes and in cortical osteocytes, which were situated within the first lamella at the endosteal side of the shaft (Fig. 1G). The endocortical osteocytes, which were located within the deeper lamellae (Fig. 1G), and the periosteal osteocytes (Fig. 1D) did not express IGFBP-2 mRNA. IGFBP-2 mRNA was also expressed in the intracortical endothelial cells of blood vessels (Fig. 1G) and in the periosteum of control tibiae (Fig. 1D). Some cells of the bone marrow, i.e., megakaryocytes, macrophages, and myeloid cells, also expressed IGFBP-2 mRNA (Fig. 1E). No differences of IGFBP-2 mRNA expression between the right and left tibiae of the control group were observed using semiquantitative analysis.
Fig. 1Expression of IGFBP-2 mRNA in rat brain sections (A, B) and in longitudinal control (C-G), sham-loaded (H), and loaded (J) tibial sections of 12-week-old female Wistar rats. The RNA signal is shown as a blue precipitate. The sections were counterstained with nuclear fast red and visualized under brightfield illumination. The scale bars represent 100 μm (A, B), 50 μm (D, E, G-J), and 100 μm (C, F). (A) IGFBP-2 mRNA expression within the choroid plexus of a control rat brain; red blood cells are negative and do not express IGFBP-2 mRNA. (B) Hybridization with representative IGFBP-2 sense probe showed no signal in the choroid plexus. (C) IGFBP-2 mRNA expression in the chondrocytes of the growth plate and osteoblasts of the trabecular bone. (D) IGFBP-2 mRNA expression in the periosteum of a control tibia; the osteocytes in the periosteal side of the shaft do not express IGFBP-2 mRNA. (E) IGFBP-2 mRNA expression in osteoblasts situated against the surface of normal trabecular bone and IGFBP-2-positive bone marrow cells. (F) Hybridization with a corresponding IGFBP-2 sense probe showed no signal in the osteoblasts, chondrocytes, and bone marrow cells of a control tibia. (G) IGFBP-2 mRNA expression within the superficial endocortical osteocytes (indicated by arrowheads), osteoblasts, and intracortical endothelial cells of a blood vessel of a control tibia. (H) IGFBP-2 mRNA expression in the endocortical osteocytes within multiple layers (indicated by arrowheads) and osteoblasts of a sham-loaded tibia. (J) IGFBP-2 mRNA expression in the endocortical osteocytes within multiple layers (indicated by arrowheads) and osteoblasts of a loaded tibia.
Control hybridizations with the corresponding sense RNA probe exhibited no signals in the brain (Fig. 1B) and tibia (Fig. 1F).
Effect of Mechanical Loading on IGFBP-2 mRNA Expression in Osteocytes of the Tibial Shaft
Induced IGFBP-2 mRNA expression was observed in osteocytes within the first lamella and within multiple layers at the endosteal side of the shaft of loaded tibia (1.68-fold increase, n = 5) (Fig. 1J) and sham-loaded tibia (1.35-fold increase, n = 5) (Fig. 1H) in contrast to the control tibia (n = 10), where IGFBP-2 mRNA expression was only seen within the first lamella of the endosteal side of the shaft (Fig. 1G).
The proportion of IGFBP-2 mRNA-positive osteocytes was 25.5 ± 12.6% (mean ± standard deviation [SD], n = 5) for loaded tibiae and 17.2 ± 10.7% (mean ± SD, n = 5) for the contralateral control tibiae (Figs. 1G, J and 2A). Sham-loaded tibiae and the contralateral control tibiae showed a proportion of IGFBP-2 mRNA-positive osteocytes of 20.5 ± 7.5% (mean ± SD, n = 5) and 13.2 ± 4.2% (mean ± SD, n = 5), respectively (Figs. 1H and 2B). Mechanical loading significantly increased the number of IGFBP-2 mRNA-synthesizing osteocytes in loaded tibiae (P = 0.001) and in sham-loaded tibiae (P = 0.031) (Fig. 2A, B). No statistical differences of IGFBP-2 mRNA-positive osteocytes were observed between the contralateral controls of the loaded and sham-loaded tibiae (P = 0.475) or between the loaded and sham-loaded tibiae (P = 0.468).
Fig. 2Influence of mechanical loading on IGFBP-2 mRNA expression in osteocytes at the endosteal side of the shaft within individual rat tibiae. Values are expressed as (A) percentage IGFBP-2 mRNA-positive osteocytes in control versus contralateral loaded tibiae (**P = 0.001, n = 5) and (B) percentage IGFBP-2 mRNA positive osteocytes in control versus contralateral sham-loaded tibiae (*P = 0.031, n = 5). No statistical differences of IGFBP-2 mRNA-positive osteocytes were observed between the control load and control sham groups (P = 0.475) or between the load and sham groups (P = 0.468). The lines connect the left and right tibiae of the same rat. The mean values of each group are represented by a bar.
Effects of Mechanical Loading on IGFBP-2 mRNA Expression in Osteoblasts, Chondrocytes, and Bone Marrow Cells
No differences in IGFBP-2 mRNA expression between loaded, sham-loaded, and control tibiae were observed in the osteoblasts, chondrocytes, and bone marrow cells by semiquantitative screening. No differences in morphology of the cells were observed between groups.
Discussion
This in vivo study showed an increase in the number of endocortical osteocytes expressing IGFBP-2 mRNA in loaded (1.68-fold) and sham-loaded (1.35-fold) tibiae 6 hours after a single period of mechanical loading with the four-point bending system. The upregulation of IGFBP-2 mRNA expression was restricted to osteocytes at the endosteal side of the shaft and multiple inner lamellae. No difference between IGFBP-2 mRNA expression in the left and right control tibia was observed. We conclude that bending and squeezing of rat tibiae result in an increase of locally synthesized IGFBP-2 mRNA by endocortical osteocytes. We suggest that IGFBP-2 plays an as yet unidentified role in the lamellar bone formation process.
We suggest that the IGFBP-2-producing osteocytes of the loaded tibiae are involved in the increased lamellar bone formation after mechanical loading. Forwood and colleagues reported that a single short period of loading using the four-point bending system resulted in an increased lamellar bone formation rate at the endosteal surface of rat tibia [25]. This has been confirmed in a validation study at our laboratory [27]. The lamellar bone formation is located at the endosteal side of the shaft, which is identical to the location of newly synthesized IGFBP-2 mRNA after mechanical loading, as demonstrated in this study. IGFBP-2 mRNA expression was restricted to this area and was not observed in the osteocytes at the periosteal side of the shaft. Mechanical loading with an external bending load of 60 N in vivo will result in an increased woven bone formation rate at the periosteal surface as a result of irritation of the periosteum and increased lamellar bone formation rate at the endosteal surface [1]. We suggest that IGFBP-2 mRNA may be less involved in woven bone formation because the osteocytes at the periosteal side of the shaft did not express IGFBP-2. This suggests that IGFBP-2 mRNA in the mechanosensitive osteocytes is specifically important for lamellar bone formation.
In addition, this study showed that the sham-loaded tibiae have increased IGFBP-2 mRNA production in endocortical osteocytes 6 hours after sham loading. Apparently, besides bending, squeezing alone is also responsible for the upregulation of IGFBP-2 mRNA in the endocortical osteocytes. However, squeezing did not induce lamellar bone formation.
Remarkably, there is a large range in the data for the percentage of IGFBP-2 mRNA-positive osteocytes in the contralateral control tibiae of the load group in comparison to the data of the contralateral tibiae of the sham group. This could be caused by the biological variation between the individuals because the animals were randomly divided between the groups and there is no significant statistical difference between those tibiae.
For this study we used the four-point bending model of Forwood because this apparatus produces a controlled mechanical strain in the tibia of living rats. An advantage of this approach is that it does not require surgical intervention and allows normal physical activity after the loading session [1, 7]. The osteogenic response will occur at a distinct location in bone. Therefore, we used the in situ hybridization technique in order to detect the local osteogenic response at the cellular level. Nonradioactive in situ hybridization is a powerful and sensitive technique to localize gene expression within decalcified rat tibiae. It was demonstrated that the IGFBP-2 probe is specific because IGFBP-2 mRNA expression is present in the choroid plexus and the neurons of the medulla oblongata, as described earlier [28], and in the chondrocytes of the proliferative and hypertrophic zone of the growth plate [34].
Nevertheless, this study has some limitations. The applied load of 60 N is supraphysiological, and 6 hours after a single loading session is too early to demonstrate bone formation.
In summary, this study shows an increase of the number of osteocytes at the endosteal side of the shaft and inner lamellae expressing IGFBP-2 mRNA 6 hours after mechanical loading and sham loading in vivo. We conclude that these IGFBP-2-producing osteocytes in the endosteal shaft are mechanosensitive. We suggest that IGFBP-2 plays a role in the lamellar bone formation process.

in vivo
mechanical loading
igfbp-2 mrna expression
nonradioactive in situ hybridization
four-point bending system


Formation of gutingimycin: analytical investigation of trioxacarcin A-mediated alkylation of dsDNA

Formation and fragmentation of recognition complexes between trioxacarcin A and various DNA sequences were examined by temperature-dependent UV and CD spectroscopy, HPLC analysis, and ESI mass spectrometry with regard to reaction conditions, intermediates, products, mechanism, and sequence specificity. Cleavage of the trioxacarcin–DNA complexes provided the natural product gutingimycin by guanine abstraction. The resulting DNA with an abasic site was further cleaved into a DNA fragment with a furanyl unit at the 3′-end and an oligonucleotide with a phosphorylated 5′-end.
Introduction
Trioxacarcins are complex natural antitumor antibiotics with anti-malaria activity [1–3]. The first trioxacarcins were isolated from streptomycetes in 1981 by Tomita [1, 2] and their structure was elucidated by Shirahata [4]. Gutingimycin (1) was isolated in 2004 from a marine streptomycete together with further trioxacarcin derivatives and named according to the ancient name of Göttingen [3, 5, 6]. It turned out to be an interesting natural product because of its mode of formation. The structure of gutingimycin (1), elucidated by X-ray analysis, suggested that gutingimycin (1) results from nucleophilic attack of the N7 atom of a guanine subunit in DNA at the epoxide of trioxacarin A (2) [5]. Natural antibiotics like hedamycin and altromycin B are able to intercalate into the dsDNA followed by alkylation of guanine at the N7 position because of their epoxide functionality [7–9]. Like hedamycin, altromycin B or kapurimycin A3 [10, 11], trioxacarcin A (2) contains an epoxide. Therefore, it has been proposed that gutingimycin (1) is not built up from low-molecular-weight precursors [5]. As for other cytotoxic natural products, it is likely that alkylation of trioxacarin A (2) is followed by depurination and DNA strand scission at the abasic site [7, 12–14]. Saito obtained derivatives structurally related to gutingimycin (1) by reaction of kapurimycin A3 with various oligonucleotides. At 0 °C a stable ionic intermediate is formed; on heating to 90 °C this dissociates into a depurinated oligonucleotide and a kapurimycin derivative [15, 16]. Hedamycin binds to dsDNA by intercalation and alkylation with a distinct preference for the central guanine in the sequence d(CGT) or d(CGG) [8, 17, 18]. The covalent hedamycin-DNA adduct was isolated by Sheil and characterized by ESI MS [19, 20].
From previous studies it was known, that trioxacarcin A (2) also forms a stable complex with dsDNA, but did not react with deoxyguanosine even at higher temperatures [1]. The aim of this study was to investigate the conditions and mechanism of the trioxacarcin-mediated alkylation of dsDNA by applying high-performance liquid chromatography and high-resolution electrospray mass spectrometry together with temperature-dependent UV and CD spectroscopy. In particular, the influence of the DNA sequence on binding and adduct formation and the identification of intermediates and cleavage products like gutingimycin (1) and DNA fragments is described in detail.
Experimental
Methods and materials
The DNA oligomers d(AATTACGTAATT) (4a), d(CAATTATAATTG) (4b), d(AATTXGXAATT) (5a–d), and the complementary oligonucleotides d(AATTGGGAATT) (7) and d(TTAACCCTTAA) (8) were purchased from IBA (Göttingen, Germany). An oligomer length of eleven or twelve nucleotides was sufficient to provide stability at room temperature (Tm = 22–35 °C). Trioxacarcin A (2) and trioxacarcin B (3) were obtained by fermentation of Streptomyces sp. isolate B8652 [6]. The reactions were carried out in aqueous phosphate buffer (10 mmol L−1 Na2HPO4/NaH2PO4, 0.1 mol L−1 NaCl, H2O, pH 7.0) with 5 μmol L−1 dsDNA and trioxacarcin A (2) in a 1:2 ratio.
UV spectroscopy
Melting curves were measured on a Perkin–Elmer Lambda 10 UV–visible spectrophotometer in connection with a Perkin–Elmer PTP-1 Peltier system following the temperature program: 0 °C (15 min) → 90 °C (180 min) → 0 °C (180 min). dsDNA (30 nmol) was dissolved in 1.5 mL phosphate buffer (10 mmol L−1 Na2HPO4/NaH2PO4, 0.1 mol L−1 NaCl, H2O, pH 7.0), trioxacarcin A (2) (60 nmol) in 1.5 mL phosphate buffer was added and the measurement was performed at a wavelength of λ = 260 nm in a quartz cuvette (1 cm).
CD spectroscopy
CD spectra were obtained on a Jasco J-810 spectropolarimeter at room temperature (data mode: CD, band width 1.0 nm, slit width: auto, sensitivity: 5 mdeg, time const.: 4.0 s; step resolution: 0.1 nm, scan speed: 50 nm min−1, five accumulations). dsDNA (30 nmol) was dissolved in 1.5 mL phosphate buffer (10 mmol L−1 Na2HPO4/NaH2PO4, 0.1 mol L−1 NaCl, H2O, pH 7.0), trioxacarcin A (2) (60 nmol) in 1.5 mL phosphate buffer was added and the measurement was performed in a quartz cuvette (1 cm).
Kinetic measurements
A solution of trioxacarcin–DNA (30 nmol) adduct in phosphate buffer (600 μL, 10 mmol L−1 Na2HPO4/NaH2PO4, 0.1 mol L−1 NaCl, H2O, pH 7.0) was heated to 80 °C for 4 h. Samples of 60 μL were taken after 0, 15, 30, 45, 60, 75, 90, 120, and 240 min and analyzed by HPLC. The area under the curve of the trioxacarcin–DNA adduct peak was plotted versus time to determine the time constant as the slope of the regression line.
High-performance liquid chromatography
HPLC separations were performed with an Äkta purifier from Amersham Pharmacia Biotech, a UV900 UV detector operated at 260 nm, a NewGuard RP-8 Brownlee Precolumn (15 × 3.2 mm, 7 μm), and an Aquapore RP-300 Brownlee Column (220 × 4.6 mm, 7 μm) from Perkin–Elmer. Samples were eluted within 45 min with a flow rate of 1 mL min−1 by applying a two-stage gradient (5–20% B in 20 min and 20–45% B in 25 min). Eluent A: 0.1 mol L−1 triethylammonium acetate buffer (H2O, pH 7.0). Eluent B: 0.1 mol L−1 triethylammonium acetate buffer (80% acetonitrile and 20% water, pH 7.0).
Electrospray mass spectrometry
To a solution of 120 nmol dsDNA in phosphate buffer (10 mmol L−1 Na2HPO4/NaH2PO4, 0.1 mol L−1 NaCl, H2O, pH 7.0) a fivefold excess of trioxacarcin A (2) was added. The resulting mixture was incubated several hours at 0 °C and the trioxacarcin–DNA adduct formed was isolated by HPLC. High-resolution mass spectrometry was performed using an Apex IV 7 T FTICR MS (Bruker Daltonics, Billerica, MA, USA) equipped with an Apollo electrospray ion source which was used in the negative ion mode (capillary voltage 4.2 kV, end plate voltage 3.8 kV). Analytes were dissolved in a water–methanol (50:50) solution and introduced by a syringe pump at a flow rate of 2 μL min−1. Nitrogen was used as drying gas at 100 °C. Hexapole accumulation was set between 0.2 s to 3.0 s and mass spectra were acquired within the mass range m/z 400 to 2000 with a resolution of about 100,000 at m/z 600. The capillary exit voltage was −80 V, −100 V, −140 V, or −170 V. Deconvolution and mass calculation for the corresponding ions were done using Xmass 6.1.2 from Bruker.
Results and discussion
In order to investigate the sequence dependency of guanine abstraction from dsDNA by trioxacarcin A (2), the two self complementary DNA oligomers d(AATTACGTAATT) (4a) and d(CAATTATAATTG) (4b) were investigated with the reactive guanine located in the center or at the end of the double strand. The position of guanine was varied to determine the influence of the helix topology on alkylation. Four additional non-self complementary oligonucleotides with the general sequence d(AATTXGXAATT) (5a–d) together with the corresponding complementary strands were examined with respect to sequence specificity. The sequences were limited to only one guanine in the double strand in order to limit alkylation to a single site. It was placed in the central position investigating all possible alterations of the neighboring nucleobases using adenine and thymine.
Fig. 1Gutingimycin (1), trioxacarcin A (2), and trioxacarcin B (3)
A first approximation of the interaction of trioxacarcin A (2) with double-stranded DNA can be obtained from temperature dependent UV-spectroscopic measurements of double-strand stabilities, because double-strand recognition, formation of a covalent adduct, nucleobase excision, or strand scission are expected to affect DNA stability. A mixture of double-stranded oligonucleotide d(AATTACGTAATT) (4a) with two equivalents trioxacarcin A (2) gave a stability of Tm = 41 °C which is significantly higher than the double strand transition of oligomer 4a without addition of the natural product (Tm = 34 °C) (Fig. 2) indicating a DNA interaction with the trioxacarcin A (2). For this interaction intercalation and groove binding of the aglycon is more likely than covalent linkage of 2, because double-strand stability obtained for d(AATTACGTAATT) (4a) with trioxacarcin B (3) is similarly increased (Tm = 42 °C) even though trioxacarcin B (3) is not able to alkylate DNA. In the cooling cycle the UV melting curve of the 4a/2 mixture provided only a stability of Tm = 28 °C. This destabilization is most likely because of nucleobase excision initiated at higher temperatures to yield gutingimycin (1) and a DNA double strand with apurine site which might further lead to DNA strand scission.
Fig. 2UV-melting curve of d(AATTACGTAATT) (4a) with and without trioxacarcin A (2) (5 μmol L−1, 1:2)
In contrast, for the oligomer with the terminal guanine 4b the differences in double-strand stability were not significant comparing the self pairing (Tm = 31 °C) with the 1:2 mixture of oligomer 4b and natural product 2 in the heating (Tm = 29 °C) or cooling cycle (Tm = 29 °C) (Fig. 3). Apparently, for DNA recognition of trioxacarcin A (2) the double helix topology is required which is not sufficiently assured at the terminus of the helix. Therefore, the guanine placed at the terminal position cannot act as a nucleophile without the help of helix recognition. This is in agreement with guanosine itself not being a nucleophile for trioxacarcin A (2) [1].
Fig. 3UV-melting curve of d(CAATTATAATTG) (4b) with and without trioxacarcin A (2) (5 μmol L−1, 1:2)
The modification of DNA 4a by trioxacarcin A (2) was also indicated by CD spectroscopy (Fig. 4). The spectra of 4a and the mixture of 4a with 2 measured at room temperature are quite similar, whereas the CD spectrum of the 4a/2 mixture heated to 90 °C differed by a shift and the intensity of the Cotton effect around 270, 250, and 220 nm.
Fig. 4CD-spectra of self pairing d(AATTACGTAATT) (4a) with (grey) and without (black) trioxacarcin A (2) (5 μmol L−1, 1:2). The dashed line indicates the spectrum obtained after interim heating to 90 °C
For a more detailed picture of trioxacarcin A (2) adduct formation with dsDNA, high-performance liquid chromatography was applied determining the components present after mixing at room temperature and after heating to 90 °C. The HPLC chromatogram of the mixture of oligonucleotide 4a with trioxacarcin A (2) before starting the heating cycle provided three peaks (Fig. 5a). Next to DNA (4a) and trioxacarcin A (2), an additional compound was indicated which was assigned to the stable covalent complex between the oligonucleotide and the natural product trioxacarcin A (4a + 2). The compound corresponding to peak (4a + 2) was isolated and characterized by high-resolution ESI MS as the DNA–trioxacarcin A adduct. After heating to 90 °C additional peaks appeared in the oligonucleotide area together with gutingimycin (1) and trioxacarcin B (3) generated by epoxide ring opening of trioxacarcin A (2) with water (Fig. 5b). The assignment of all HPLC peaks was confirmed by co-injection and mass spectrometry.
Fig. 5HPLC chromatogram of a mixture of oligonucleotide 4a and trioxacarcin A (2): a at room temperature; b after heating to 90 °C; next to DNA 4a further oligonucleotides and oligonucleotide fragments are eluting between 11 and 17 min
Examining the trioxacarcin A (2) interaction with oligomer d(CAATTATAATTG) (4b) carrying the guanine at the terminal position, the HPLC chromatogram turned out to be quite similar to the HPLC chromatogram obtained after interaction with DNA 4a. Nevertheless, the yield of the respective DNA–trioxacarcin adduct (4b + 2) was significantly lower and gutingimycin (1) was only obtained in traces after heating (Fig. 6). As indicated in Table 1, adduct formation of trioxacarcin A (2) with oligonucleotide 4a is about five times more efficient than binding to DNA 4b. Sterically, the nucleophilic attack should be easier at the end of the double helix, but the ease of adduct formation turned out to be higher in the central position of the double strand. Therefore, trioxacarcin A (2) seems to take advantage of dsDNA recognition by intercalation or groove binding prior to nucleophilic attack. This recognition behavior is also described for the natural products hedamycin and altromycin B of the pluramycin family [7, 8, 21, 22].
Fig. 6HPLC chromatogram of a mixture of the oligonucleotide 4b and trioxacarcin A (2): a at room temperature; b after heating to 90 °CTable 1Examined oligomers and adduct formationDNA oligomers Adduct formula[Madduct − 6H]6− a calcd. / found mass[Madduct + Na − 7H]6− a calcd. / found massAdduct (%)bAATTACGTAATT4aC161H202N43O90P11752.32 / 752.33755.99 / 755.9953CAATTATAATTG4bC161H202N43O90P11752.32 / 752.39755.99 / 756.0010AATTAGTAATT5aC152H190N40O84P10704.15 / 704.16707.81 / 707.8313AATTTGAAATT5bC152H190N40O84P10704.15 / 704.16707.81 / 707.825AATTTGTAATT5cC152H191N37O86P10702.65 / 702.65706.31 / 706.3116AATTAGAAATT5dC152H189N43O82P10705.65 / 705.66709.31 / 709.321a Adduct ions up to the sevenfold charged ion were observed; the relative intensities varied from oligomer to oligomerb Conversion rate given by the trioxacarcin A to adduct proportion
The isolated adduct of oligonucleotide 4a with trioxacarcin A (2) is stable at room temperature for more than 24 h and at 56 °C for at least 4 h. At 80 °C within 4 h, decay to gutingimycin (1) was observed obeying first-order kinetics (Fig. 7).
Fig. 7Kinetic analysis based on a decay of the d(AATTACGTAATT)–trioxacarcin A adduct (4a + 2) [23]
In order to get preliminary information about sequence selectivity in trioxacarcin A (2) recognition and alkylation, all adenine and thymine combinations were investigated as the neighboring nucleobases of a central guanine. The four oligonucleotides 5a–d reacted with trioxacarcin A (2) to yield the corresponding adducts, which were characterized by high-resolution ESI MS. The corresponding HPLC profiles of oligomers 5a–d with trioxacarcin A (2) before and after heating to 90 °C were almost similar, except for the peak height of the generated adducts. The conversion rate was calculated from the peak area proportion of trioxacarcin A (2) to the adduct. As indicated in Table 1, the conversion rate from trioxacarcin A (2) to the corresponding DNA adducts varied from 1 to 16%. Based on non-self complementary double strands 5a–d and results obtained by HPLC and ESI MS, adduct formation seems to be preferred when thymine is placed next to guanine with thymine neighboring the 3′-site having a greater impact. It still needs to be investigated if this result holds only for thymine in comparison with adenine or if this is a more general preference for pyrimidines over purines neighboring the trioxacarcin-substituted guanine.
The alkylation of oligomers with more than one guanine was investigated with the double strand formed by the complementary strands d(AATTGGGAATT) (6) and d(TTAACCCTTAA) (7) which provide a quite stable double strand (Tm = 35 °C). In a mixture of 6 + 7 with six equivalents of trioxacarcin A (2) the double strand stability decreased to Tm = 29 °C (heating cycle) whereas double strand formation was no longer detected after heating to 90 °C. This leads to the conclusion that trioxacarcin A (2) addition and DNA degradation is accelerated with three neighboring guanines. The high tendency for adduct formation of trioxacarcin A (2) to dsDNA 6 + 7 at 0 °C was indicated by an HPLC signal at 26.0 min nearly the size of the remaining oligomer 7 (Fig. 8a). Even after 24 h signals appeared at 30.8 and 31.6 min that were assigned to DNA 6 functionalized with two equivalents of trioxacarcin A (2). The amount of double substitution increased significantly within 72 h reaction time (Fig. 8b).
Fig. 8HPLC chromatograms of a mixture of the DNA double strand d(AATTGGGAATT) (6) + d(TTAACCCTTAA) (7) and trioxacarcin A (2): a 24 h at 0 °C and b 72 h at 0 °C
Further insight into the trioxacarcin A (2)-initiated substitution reaction and the DNA cleavage products were obtained by additional ESI MS experiments stepwise increasing the capillary-skimmer dissociation energy in the ion source [24–30]. Like increasing the temperature this also initiated fragmentation of alkylated DNA followed by strand cleavage. The adduct of trioxacarcin A (2) with oligomer 5d was unequivocally assigned by ESI MS with a low capillary exit voltage of −80 V providing exclusively ions of the DNA–trioxacarcin complex 5d + 2 (Fig. 9a): ([M − 3H]3−, [M − 4H]4−, [M − 5H]5−, and [M − 6H]6−). By applying capillary–skimmer dissociation by raising the capillary exit voltage to −140 V the signals for the DNA–trioxacarcin A adduct were also found (Fig. 9c). Nevertheless, the adduct ions were less intense. One of the peaks with the highest intensity appears at m/z 1026.37; this is assigned to the single negatively charged ion of gutingimycin (1) comparable to the thermal cleavage product observed already by HPLC. Depurination is known to result in oligonucleotide strand scission [19, 20, 27]. Furthermore, it has been reported that collision-activated dissociation of negatively charged oligonucleotide ions starts with cleavage of a base followed by cleavage of the 3′ C–O bond of the sugar unit from which the base was lost [31]. In accordance, two ESI MS peaks were correlated to the doubly charged w5-fragment ion at m/z 781.64 (calculated 781.63) and the doubly charged (a6-B)-type fragment ion at m/z 821.66 (calculated 821.64) of the oligonucleotide 5d. The accompanying smaller peaks to the right of w52− and [a6-GT]2− were assigned to the corresponding [M + Na − 3H]2− ions. These fragments might mechanistically be explained by the initiation of strand cleavage at C3′ leading to a furanyl unit at the 3′-end of d(AATTA) and a phosphoryl group at the 5′-end of the second fragment d(AAATT) [31]. An additional small peak m/z 801.90 [10 − 4H]4− can be assigned to the depurinated oligonucleotide 10 (Scheme 1, together with its sodium peak m/z 807.40 [10 + Na − 5H]4−). Nevertheless, oligonucleotide 10 with a double bond between C1′ and C2′ (Scheme 1) has a calculated mass [10 − 4H]4− of m/z 801.89 and [10 + Na − 5H]4− of m/z 807.38 corresponding to the mass of the intermediate fragment. This assignment is clearly supported by the corresponding deconvoluted mass spectra (Figs. 9b and d).
Fig. 9a ESI mass spectrum of trioxacarin-DNA d(AATTAGTAAATT) 5d + trioxacarcin (2) adduct, capillary exit potential −80 V; b Respective deconvoluted spectrum of trioxacarin-DNA 5d adduct; c ESI mass spectrum of trioxacarin-DNA d(AATTAGTAAATT) 5d + trioxacarcin (2) adduct, capillary exit potential −140 V; d Respective deconvoluted spectrum of trioxacarin-DNA 5d adductScheme 1Proposed cleavage reaction of DNA–trioxacarcin A adduct
The fragments observed for depurination and DNA strand scission of trioxacarcin–DNA adducts are in agreement with the results from Sheil reporting similar oligonucleotide fragmentation in ESI MS–MS experiments for various hedamycin–DNA adducts [19]. Based on the ESI MS fragments detected by cleavage of the trioxacarcin–DNA adduct (5d + 2) the following products and intermediates for the fragmentation of the trioxacarcin–DNA adduct are proposed (Scheme 1): Depurination is generating gutingimycin (1) and the oligonucleotide with an abasic site 10 dissociating in fragment 8 with a furanyl unit at the 3′-end (w5-type fragment) and fragment 9 with a phosphorylated 5′-end (a6-B-type fragment). The phosphoester 9 seems to be a perfect leaving group for generation of a furan.
The results described for the trioxacarcin–DNA 5d adduct were supported by the mass spectra of the trioxacarcin adducts with oligonucleotides 4a–b and 5a–c. The signals assigned to adducts, abasic sites, fragments, and gutingimycin were also observed for these oligomer adducts. With low dissociation energy in the ion source (capillary exit potential −80 V) the adduct peak was obtained for the self complementary double strand 4a with guanine in the central position almost exclusively, whereas fragmentation already occurred in the case of oligomer 4b with the terminal guanine obtaining the gutingimycin signal as the most prominent. Trioxacarcin A adducts with oligonucleotides 5a–c always provided the strongest ESI MS signals for the adduct peaks using the lowest dissociation energy; nevertheless, the peak intensities corresponding to the other fragments varied. The trioxacarcin adducts with oligomers 5a and 5d were the most stable.
Conclusions
The formation of stable adducts of the natural product trioxacarcin A (2) with guanine-containing dsDNA was investigated by a combination of temperature-dependent UV and CD spectroscopy, high-performance liquid chromatography, and high-resolution mass spectrometry. Recognition and non-covalent binding of trioxacarcin A (2) to dsDNA seems to be a prerequisite for nucleophilic attack of the guanine to form a covalent complex. Preliminary evidence indicates that trioxacarcin A-DNA interaction has sequence specificity with greater reactivity observed for guanines 5′ to thymidines. Cleavage of the trioxacarcin–DNA adducts can be initiated at higher temperatures or by applying capillary-skimmer dissociation by raising the capillary exit potential in mass spectrometry experiments leading to the formation of the bioactive secondary metabolite gutingimycin and DNA strand scission. As DNA cleavage products, a fragment with a furanyl unit at the 3′-end was identified next to an oligonucleotide with a phosphorylated 5′-end.

gutingimycin
trioxacarcin a
electrospray mass spectrometry
dna alkylation
dna strand cleavage


Pure cervical radiculopathy due to spontaneous spinal epidural haematoma (SSEH): report of a case solved conservatively

Introduction: Spontaneous spinal epidural haematoma (SSEH) is widely recognised throughout the literature as a cause of myelopathy, radicular compression being very rarely reported. Surgical management is almost always recommended, especially in the cases of spinal cord compression. Conservative treatment is reported as a curiosity and only in the case of spontaneous improvement. This report presents the particular case of a 64-year-old patient undergoing anticoagulant therapy that had a cervical radiculopathy due to a SSEH confirmed by MRI. The patient improved spontaneously and symptoms were solved with unconventional conservative treatment and without stopping the anticoagulant therapy. Conclusions: Spontaneous epidural haematoma must be kept in mind when patients undergoing anticoagulant therapy have a sudden onset of cervicobrachialgia. Even though most spinal surgeons advocate surgical treatment, a conservative approach may lead to a complete recovery and may be considered as a good option in the case of radicular involvement. Discontinuation of the anticoagulant therapy may not always be needed, especially when the clinical syndrome improves spontaneously.
Introduction
Although spontaneous spinal epidural haematoma (SSEH) is a low incidence condition, it is widely recognised throughout the literature as a cause of myelopathy [5, 15]. The relationship between SSEH and anticoagulant therapy is well known and the probable cause of bleeding is thought to be the rupture of the venous epidural plexus during a sudden elevation of thoracic or abdominal pressure [4, 6, 8, 10, 11, 13, 15].
The most common symptoms are cervical or thoracic pain followed by cord compression signs. Radicular involvement is very rare and, when it appears, it mostly affects the lumbar spine, producing ciatalgia [1, 3, 11]. Although the currently suggested approach is an urgent surgical decompression [9, 10], conservative treatment is recommended when there is an objective improvement of the neurological status [2, 11, 12]. In general, anticoagulation treatment must be suspended or adjusted before surgery.
The originality of this report arises in three points:The case involved the cervical spine producing simple radiculopathy.It was solved spontaneously.Anticoagulation therapy was not discontinued.
Case report
A 64-year-old white man undergoing anticoagulant therapy because of cardiac valve prosthesis arrived at the emergency room of our Hospital suffering from a sudden onset of cervical pain and a left C5 brachial pain and weakness. The physical exam revealed a 4/5 deltoid weakness and abolition of the bicipital reflex. The patient showed no signs or symptoms of cord compression. Standard radiographs of the cervical spine were normal.
Based on the suspicion that it was a case of disc herniation with left C5 root involvement, symptomatic treatment by means of non-steroid anti-inflammatory drugs and painkillers was indicated. A cervical MRI was scheduled and the patient was invited to undergo further examination at the Spinal Surgery Unit. Owing to the fact that the first physician was not a spinal surgeon and therefore not aware of the relationship between anticoagulation and SSEH, discontinuation of anticoagulant therapy was not indicated. Two days later, when the patient was examined by the Spinal Surgery Unit, the pain had been completely relieved and the weakness had also decreased. The MRI revealed a left posterolateral ovoid mass compatible with a haematoma extending from C4 to C5 (Fig. 1). Symptomatic medical treatment was interrupted. Once the specialist knew the real cause of the radicular syndrome, the haematologist was consulted and they preferred not to suspend the anticoagulant therapy (despite an adequate level of anticoagulation, INR 2,7) in order to prevent thromboembolism.
Fig. 1MRI 2 days after the onset of symptoms. Posterolateral placement of the haematoma at C4–C5 level. a, c Isointense appearance of the haematoma on the MRI T1-weighted sagittal and axial images. b, d Hyperintense appearance of the haematoma on the MRI T2-weighted sagittal and axial images
Seven days later, the patient was free of weakness and the MRI showed a decrease in the haematoma size (Fig. 2). Successive MRI results were obtained 1 month (Fig. 3) and 1 year later on (Fig. 4) to document the resolution of the haematoma. One year on, the patient remains asymptomatic.Fig. 2MRI performed 7 days later. The haematoma is smaller nevertheless it is located at the same C4–C5 levelFig. 3MRI 1 month later: disappearance of the haematomaFig. 4MRI 1 year later: normal MRI
Discussion
Radicular compression due to SSEH is far less frequent than myelopathy. Groen and Van Alphen [6] reported that 4.5% of a series of 320 SSEH cases treated surgically presented radiculopathy; all cases affecting the lumbar spine. Recently, Groen [7] reported that 9% of a series of 64 SSEH cases treated conservatively presented an isolated radicular compromise; only one of his cases was located on the cervico–thoracic spine. Our case adds little in terms of number but the fact that it may be the only report of its kind on purely cervical radiculopathy due to SSEH, which may be considered anecdotic, as well as interesting.
Although the spreading of the haematoma throughout the epidural space is the most likely hypothesis proposed for spontaneous recovery in case of neurological impairment [7, 14], our case does not support that theory because the haematoma did not spread throughout the epidural space. (Figs. 1, 2)
Conservative management is currently indicated in uncommon situations or when neurological symptoms improve before medical evaluation. Geographic isolation, initial inaccurate diagnosis, neurological improvement pending an adequate coagulation level prior to surgery, high surgical risks and several other reasons had been referred to as the likely causes that led to the opportunity for spontaneous recovery [4, 7, 9, 11, 16]. Our case can be categorised as wrong diagnosis initially, but we have to admit that the first non-specialised consultation spared our patient from the operation. If the patient had first consulted a more skilled specialist, following the state of the art, he would have probably been operated on, thus obtaining a good result and a proud surgeon.
Finally, the main interest of our case would be focused on the controlled maintenance of the anticoagulant therapy in patients with mild neurological compromise such as radiculopathy due to SSEH in order to avoid thromboembolic risk.

conservative treatment
anticoagulant therapy
cervicobrachialgia
spontaneous spinal epidural hematoma


Invasive carcinomas of the male breast: a morphologic study of the distribution of histologic subtypes and metastatic patterns in 778 cases

The current investigation was conducted to evaluate the proportional distribution of the various histologic subtypes (including newly recognized variants) of male breast carcinomas, to determine whether any histologic subtypes occur with a frequency that is markedly discordant with the expected frequencies from published data on parallel female breast tumors. We also aimed to document the distribution of malignancies metastatic to the breast. Seven hundred fifty-nine archived cases of primary invasive carcinoma involving the male breast were retrieved and subcategorized into histologic subtypes according to contemporary criteria. Six hundred forty-three (84.7%) tumors were pure infiltrating ductal carcinoma (IDC) not otherwise specified. The most common of the remainder included papillary carcinoma with invasion in the form of IDC (n = 34), mixed IDC and mucinous carcinoma (n = 26), and pure mucinous carcinoma (n = 21). In 19 cases, metastases from other sites involved the breast, most commonly (58%) cutaneous melanoma. Invasive carcinoma of the male breast appears to display a morphologic spectrum and distribution of histologic subtypes that is comparable to those of the female breast, with some expected variation. Compared with published experience on their female counterparts, there is a two-fold increase in the frequency of invasive papillary carcinoma in the male breast. Finally, the most common tumor metastatic to the male breast in this series was cutaneous melanoma.
Introduction
Breast cancer in males is relatively uncommon, with approximately 1,450 newly diagnosed cases annually in the United States, and an associated mortality rate that accounts for less than 0.2% of all cancer-related deaths in men [1, 4, 9, 15, 17, 20, 28]. However, recent epidemiologic studies suggest that the incidence of male breast cancer has been steadily increasing [7, 14]. Utilizing data from the National Cancer Institute’s Surveillance, Epidemiology and End Results database for the 26-year period between 1973 and 1998, Giordano et al. [7] found that the incidence of male breast cancer increased from 0.86 to 1.08 per 100,000 population. This increasing incidence, in addition to the better elucidation of the role of BRCA2 mutations in male breast cancer, has renewed interest in specifically identifying the underlying pathogenesis of male breast cancer and clarifying the specific differences, if any, between male and female breast cancer.
On a broad demographical level, male and female breast cancers are fundamentally similar with the main differences lying in older age of occurrence and lower incidence of the former. However, significant differences have been noted between male and female breast cancers with respect to the expression of a variety of biologic factors, including hormone receptors such as estrogen receptor, progesterone receptor, c-erbB-2, estrogen-inducible proteins such as pS2, Cathepsin D, hsp27, proteins related to basement membrane and extracellular matrix degradation such as the urokinase system of plasminogen activation and their inhibitors, and protooncogenes such as bcl-2 [2, 6, 7, 18, 21, 22, 24, 27]. At the morphologic level, male ductal intraepithelial neoplasia (ductal carcinoma in situ), in contrast to similar lesions in females, displays a distinct histologic profile in which the majority of tumors are of the papillary type, with cribriform, micropapillary, and solid types being much less common [13]. In particular, the comedo-type of DIN (comedo DCIS) is notably rare in the male breast [13]. For invasive carcinomas, the female and male tumors are morphologically indistinguishable [26] and many published studies on male breast cancer frequently represent the major histologic subtypes of nonlobular breast carcinoma, such as ductal, medullary, mucinous, and papillary [3, 7, 8, 12, 19, 22, 25]. However, these studies generally do not have the distribution and relative frequencies of the various histologic subtypes as their main focus, and as such, pathologically important distinctions such as signet ring vs colloid carcinoma (both subsumed under mucin producing carcinoma), or metaplastic carcinoma vs infiltrating duct carcinoma not otherwise specified are not emphasized. Over- or underepresentation of a particular histologic subtype in male breast cancers may provide valuable insight into the etiopathogenetic differences between male and female breast cancers and may provide an important nidus for further studies.
In this study, we investigated the frequencies and distribution of the various histologic subtypes in a large data set of invasive carcinomas seen at a large tertiary center over a 40-year period. Our goals are to document these frequencies and distribution and to see whether any histologic subtypes are notably distinct in frequency of occurrence in the male breast, as compared with historical published data on similar lesions in the female breast. The frequencies and sites of origin of metastatic tumors to the male breast during the study period were also investigated.
Materials and methods
After approval from our institutional research review committee, cases of invasive carcinoma involving the male breast diagnosed between 1957 and 1997 were retrieved from the archived institutional files of the Armed Forces Institute of Pathology (AFIP, Washington DC, USA). The available records and slides on each patient were reviewed. All cases with ambiguous gender (even if one among the multiple entries for gender was coded as female rather than male) were excluded (n = 38). Diagnostic designations were assigned according to contemporary WHO criteria [23]. All special types of carcinoma inclusive of mucinous, tubular, secretory, adenoid cystic, lobular, metaplastic, etc. (n = 116) were reviewed extensively; many of these cases had been evaluated repeatedly as part of various previously published studies from the department and slides of good quality were available on these cases for review. Among the 643 cases of infiltrating duct carcinoma not otherwise specified, only cases with good quality and adequate representative slides (n = 365) were graded, using the modified Scarf–Bloom Richardson scoring system. On 78 cases, the slides were so old that we could only confirm the presence of a ductal type carcinoma. The other 200 cases did not have all of the patients’ slides pertaining to the lesion available and the final interpretation was based on the descriptions of morphology in the AFIP files. Because cases sent to AFIP are mostly for confirmation of diagnosis, information about therapy beyond what was in the surgical pathology report or the accompanying cover letter are generally not available. Some patients had repeated material sent to AFIP as the lesion recurred, which is how follow-up information was collected in a small subset of cases.
Information about age, operative procedure, nodal excision, and distant metastases were retrieved from the patients’ AFIP files. Diagnostic designations were assigned largely based on morphologic and not immunohistochemical features since in many cases there were no blocks or unstained slides available for additional studies. Thirty of these cases were the subject of a previous report [13], which focused on in situ carcinomas and were reported in 1998. Another 113 cases, 8 of which were ductal in situ and another 9 papillary carcinomas, were subject of another report in 1969 [19] Cases of nonhematopoietic or lymphoreticular malignancies metastatic to the male breast during this period were also catalogued.
Results
Seven hundred and seventy-eight cases of breast carcinoma were reported as involving the male breast during the study period. Seven hundred fifty-nine of these cases were considered primary to the breast and 19 as metastatic to it. The patients ranged in age from 9 to 94 years (mean 61 years). The age distribution available for 520 cases was as follow: 8 were ≤30 years, 22 were ≤40 years, 205 were 41 to 60 years, 251 were 61 to 80 years, and 34 were over 80 years of age. Among the 490 for whom the racial information was available, 401 were Caucasian, not of Hispanic origin, 82 were Black, not of Hispanic origin, 6 were Asians or Pacific Islander, 1 was Hispanic, and the race was unknown for 269. The carcinomas ranged from ≤1 mm (microinvasive) to 12 cm. Many of the cases with multifocal early invasion of 1 to 7 mm were associated with large solitary or multifocal papillary intraductal carcinomas. The various histologic subtypes and their proportional distribution are outlined in Table 1, whereas representative images are illustrated in Fig. 1. Pure infiltrating duct carcinoma not otherwise specified was the most common histologic subtype, representing 84.7% of all cases. Six hundred forty-three cases were in the latter group, including 34 cases associated with Paget’s disease, 17 cases with prominent apocrine features in <50% of tumor volume (i.e., insufficient to qualify as apocrine carcinomas), 4 cases with prominent medullary features (tumor circumscription, cellular syncytia, sparse lymphocytic infiltrate), 2 cases of infiltrating duct carcinoma with signet ring cells (<5% of tumor volume), 3 cases of infiltrating duct carcinoma with focal clear cells (<5% of tumor volume), and 3 cases of carcinoma with osteoclast-like giant cells. Papillary intraductal carcinoma with invasion in the form of infiltrating duct carcinoma represented 4.5% (34/759 each) of all cases. Other well-represented histologic subtypes or combinations included mixed mucinous and infiltrating duct carcinoma (n = 26, 3.4%), pure mucinous carcinoma (21, 2.8%), and adenoid cystic carcinoma (n = 8,1.05%). Other variants each constituted less than 1% of all cases (Table 1). Notably, there were three cases of lobular carcinoma. As noted previously, these diagnoses were not based on immunohistochemical confirmation by immunostain for E-cadherin, but all three showed archetypal morphologic features of lobular carcinoma with loosely cohesive single cell files and rare intracytoplasmic lumens. Two of these cases had evidence of lobular development in the surrounding breast but the slides available for review did not show lobular intraepithelial neoplasia (LIN). There were also three cases of mixed lobular and ductal carcinomas, in which these areas were spatially separate. The youngest patient, a 9-year-old boy was the only one with pure secretory carcinoma. Five of 759 cases were microinvasive (invasive focus less than 1 mm in maximum size in a predominantly intraepithelial lesion); three of these five cases were multifocal, whereas the other two were unifocal. Of the 365 cases of infiltrating duct carcinoma not otherwise specified that were graded, 104 were grade 1, 137 were grade 2, and 124 were grade 3. Among the 93 with lymph nodes available for assessment, 61 (66%) cases had axillary metastases. Forty of the women with axillary node metastases also had distant metastases. The most common distant metastatic sites were lung, adrenal glands, and bone. In addition, there were 19 examples of metastases to the breast that were documented. Of these, 58% (11/19) were cutaneous melanomas, and 21% (4/19), 10% (2/19), and 10% (2/19) represented carcinomas from the prostate, lung, and larynx, respectively.
Table 1Distribution of the various histologic subtypes in 778 invasive carcinomas involving the male breastHistologic subtypeNumber of casesPercentageInfiltrating duct carcinoma not otherwise specified, includinga64384.7Carcinoma associated with Paget’s disease34Carcinoma with prominent apocrine features17Carcinoma with medullary features4Carcinoma with osteoclast-like giant cells3Carcinoma with focal signet rings2Carcinoma with focal clear cells2Pleomorphic carcinoma2Carcinoma with no notable distinctive features579Papillary intraductal carcinoma with invasion in the form of infiltrating duct carcinoma344.48Mixed infiltrating duct and mucinous carcinoma263.4Pure mucinous carcinoma212.8Adenoid cystic carcinoma81.05Tubular carcinoma60.8Small cell carcinoma60.8Invasive lobular carcinoma30.39Solid neuroendocrine carcinoma30.39Mixed lobular and ductal carcinoma30.39Adenocarcinoma with spindle cell  metaplasia20.26Pure signet ring carcinoma (no in situ component)10.13Invasive cribriform carcinoma10.13Secretory carcinoma10.13Squamous cell carcinoma10.13Total759100Metastatic lesions19Final total778aSubsets included for information only (not WHO categories)Fig. 1Representative pathologic images of the various histologic subtypes. a and b Gross and microscopic images of infiltrating duct carcinoma, the most frequently encountered histologic subtype (b original magnification ×160). c A colloid (mucinous) carcinoma (right field) occurring in a gynecomastoid breast (left field) (original magnification ×100). d The single case of a secretory carcinoma was in a 9-year-old boy, the youngest patient in this series (original magnification ×140). e and f Examples of papillary intraductal carcinoma with invasion in the form of infiltrating duct carcinoma [original magnifications: ×100 (e), ×120 (f)]
Discussion
The frequency, distribution, and morphologic spectrum of the various histologic subtypes of male breast cancer as compared with its female counterpart may provide a valuable insight into the etiology and/or pathogenesis of both. This was perhaps best illustrated in the study of ductal carcinoma in situ by Hittmair et al. [13]. The authors showed that in the male breast, papillary DCIS occurred with a much higher frequency (46% of pure lesions) than expected, while solid, micropapillary, and cribriform patterns occurred with a much lower frequency (22% of pure DCIS combined). Notably, only 5 of 114 cases were of comedo-type DCIS and only 7 of 114 cases were considered high grade. The authors concluded that predominance of papillary patterns among intraepithelial male breast lesions is a reflection of the relative abundance of ducts and the poorly developed terminal-ductal lobular units in male breast. Given the rarity of true comedo DIN in male breast, the authors speculated that the fully developed lobular environment and the female type hormonal milieu may be a requirement for development of comedo DIN [13].
In this study, we investigated in detail the frequencies and distribution of the various histologic subtypes in a large data set of invasive carcinomas seen at a large tertiary center over a 40-year period. Our findings suggest that invasive carcinoma of the male breast displays a morphologic spectrum and distribution of histologic subtypes that is comparable to invasive carcinoma of the female breast and that no specific histologic subtypes are notably over- or underrepresented with the exception of papillary and lobular variants.
Papillary intraductal carcinomas basically assumed an infiltrating duct carcinoma pattern once they invaded the stroma (Fig. 1e,f). A papillary component was present in 34 (4.5%) of 759 cases, representing the second most common histologic subtype. These papillary carcinomas appeared to have invaded either through massive expansion and total disruption of ductal confines with typical invasive patterns at their periphery or only focally disrupted the duct wall and invaded the surrounding stroma as an infiltrating duct carcinoma. The latter represents approximately 2% [5] of female breast invasive carcinomas and appears overrepresented in males. In a large population-based study, Giordano et al. [7] reported that 2.6% of invasive malignancies in males were papillary carcinomas, as compared with 0.6% in females (p < 0.0001). However, the differences between males and females with respect to the frequencies of the other histologic subtypes were also statistically significant. The significance of the papillary histologic subtype or papillary architecture in male ductal carcinoma requires further investigation but is most probably related to the architecture of the male mammary duct system with dominance of larger ducts.
The possibility of metastases to the breast should be considered when evaluating breast masses particularly if there is a history of cancer elsewhere. Nineteen cases of carcinoma metastatic to the male breast were seen over this study period, representing 2.4% (19/778) of all nonhematopoietic or lymphoreticular malignancies. This is again a twofold increase comparable to the 1.2% reported in female breast malignancies over a 10-year period [11]. Of the 19 metastatic tumors, 58% (11/19) were cutaneous melanomas, and 21% (4/19), 10% (2/19), and 10% (2/19) represented carcinomas from the prostate, lung, and larynx, respectively. It is interesting to note that prostatic carcinoma, which has been the subject of most reports of carcinomas metastatic to the male breast [10], was not the most common metastatic tumor in this series. The number of metastatic tumors in this series is relatively small, which is a limitation in interpreting the distribution of originating sites. Nonetheless, in the investigation of metastatic tumors to the male breast of an unknown primary site, our findings provide additional data on some of the most likely sites.
This study also confirms the occurrence of lobular carcinoma in the male breast. Three cases of invasive lobular carcinoma, diagnosed purely on a morphologic basis, were present among 759 cases (0.4%). Three additional cases of mixed lobular and ductal carcinoma were also diagnosed over this period (0.4%). Joshi et al. [16] found one (4%) lobular carcinoma out of 27 cases, while Goss et al. [8] found 4 (1.9%) cases out of a total of 229. In the series of Giordano et al. [7], lobular carcinomas constituted 1.5% for their male breast cancers. It is noteworthy that all of these cases were interpreted as lobular based on morphology without E-cadherin immunostain confirmation.
In summary, invasive carcinoma of the male breast appears to display a morphologic spectrum and distribution of histologic subtypes, which is comparable to invasive carcinoma of the female breast with some variation. As expected, lobular carcinoma is exceedingly rare but was encountered in this series. There appears to be a twofold increase in the frequency of invasive papillary carcinoma and metastatic cancers to the male breast as compared to published data in the female breast. Finally, the most common tumor metastatic to the male breast in this series was cutaneous melanoma.