The discovery and mechanism of action of letrozole

Because estrogen contributes to the promotion and progression of breast cancer, a greater understanding of the role of estrogen in breast cancer has led to therapeutic strategies targeting estrogen synthesis, the estrogen receptor, and intracellular signaling pathways. The enzyme aromatase catalyses the final step in estrogen biosynthesis and was identified as an attractive target for selective inhibition. Modern third-generation aromatase inhibitors (AIs) effectively block the production of estrogen without exerting effects on other steroidogenic pathways. The discovery of letrozole (Femara®) achieved the goal of discovering a highly potent and totally selective AI. Letrozole has greater potency than other AIs, including anastrozole, exemestane, formestane, and aminoglutethimide. Moreover, letrozole produces near complete inhibition of aromatase in peripheral tissues and is associated with greater suppression of estrogen than is achieved with other AIs. The potent anti-tumor effects of letrozole were demonstrated in several animal models. Studies with MCF-7Ca xenografts successfully predicted that letrozole would be clinically superior to the previous gold standard tamoxifen and also indicated that it may be more effective than other AIs. An extensive program of randomized clinical trials has demonstrated the clinical benefits of letrozole across the spectrum of hormone-responsive breast cancer in postmenopausal women.
Introduction
Studies have consistently shown that lifetime exposure to estrogens increases the risk of breast cancer [1]. The degree of risk is increased by persistently elevated blood concentrations of estrogen [2]; clinical indicators of persistently elevated blood estrogen concentrations, for example, age at menarche, first live birth, menopause, alcohol consumption, and obesity [3–5]; and, although still controversial, exposure to exogenous estrogen, for example, some forms of hormone replacement therapy and oral contraceptives [6–12]. The presence of some of these factors also increases the risk of breast cancer being estrogen receptor (ER)-positive [13]. Studies have shown that higher levels of endogenous estrogen and testosterone (which is converted to estrogen by aromatase) increases breast cancer risk, regardless of predicted breast cancer risk [14–16]. These data indicate that estrogen is an important risk factor even in women considered at high risk of developing the disease, for example, those with a family history of breast cancer.
Estrogen is thought to contribute to the initiation and contributes to the promotion and progression of breast cancer via two complementary mechanisms [1], the carcinogenic effects of estrogen metabolites, notably hydroxyl metabolites [3, 17, 18], and stimulation of ER signaling pathways, including those initiated by activation of epidermal growth factors, notably the mitogen-activated phosphoinositide 3 kinase pathway [19–30]. Greater understanding of the role of estrogen in breast cancer has led to therapeutic strategies targeting estrogen synthesis (aromatase inhibitors [AIs]) [31], the ER (selective ER modulators [SERMs], pure antagonists) [32], and intracellular signaling pathways (signal transduction inhibitors) [33].
Hormone receptor (HR)-positive tumors are defined as those with ER or progesterone receptor (PgR) expression detectable above a pre-set limit [34]. Patients whose ER or PgR expression is below this pre-set limit are considered HR–. Approximately two thirds of breast cancer patients have HR+ tumors [13] and are candidates for treatment strategies designed to counteract the growth effects of estrogen. This review describes the rational development of the potent AI letrozole, which has therapeutic utility in HR+ tumors across the breast cancer continuum.
Mechanism of action of aromatase inhibitors
Aromatase
Aromatase (cytochrome P-450 [CYP] 19) catalyzes the rate-limiting step (conversion of steroidal C-19 androgens to C-18 estrogens) in estrogen biosynthesis [35–37]. Aromatization is the final step in steroid biosynthesis (Fig. 1) [38]; and, therefore, aromatase is an attractive target for selective inhibition [39, 40]. Aromatase is expressed primarily in the ovary and also in central and peripheral tissues, fat, muscle, liver, and breast [41, 42]. With increasing age, as ovarian estrogen production declines [43], the contribution of peripheral production of estrogens increases [44], and in postmenopausal women, peripheral aromatization of androstenedione produced by the adrenal gland (Fig. 1) [38] becomes the main source of endogenous estrogens [45–49]. Of note, normal and malignant breast tissue contributes to the peripheral synthesis of estrogens [14, 50–53]. Thus, expression of aromatase in breast tumors may contribute significantly to the degree of cellular exposure to estrogens [14]; therefore, it is important to target both intra-tumoral and peripheral aromatase [31].
Fig. 1Aromatization of androgens to estrogens in postmenopausal women. A androstenedione, E1 estrone, E1S estrone sulfate, E2 estradiol, T testosterone. Reprinted from [38] with permission from the Society of Endocrinology
The presence of intracellular aromatase activity could explain why estrogen concentrations are 10–20 times higher in peripheral tissue than blood in postmenopausal but not pre-menopausal women [41, 54–58]. Moreover, estrogen concentrations are higher in tumors than in surrounding non-malignant tissue [41, 54–58]. Recent research has increased understanding of how aromatase is regulated by tissue-specific promoters [59] and how genetic variation may affect the pathophysiology of estrogen-dependent disease [60]. Pharmacogenomics may become an increasingly important tool for individualizing hormonal therapy for patients with breast cancer.
Aromatase inhibitors
Modern third-generation AIs effectively block the production of estrogen without exerting effects on other steroidogenic pathways and have been heralded as a “triumph of translational oncology” [61]. The search for potent and selective inhibitors of aromatase started with the first-generation inhibitor aminoglutethimide [62]. However, aminoglutethimide lacked selectivity for aromatase [63] and inhibited biosynthesis of cortisol, aldosterone, and thyroid hormone [64] as well as aromatase; moreover, aminoglutethimide was also found to induce hepatic enzymes (Fig. 2) [65, 66]. Second-generation AIs included the nonsteroidal inhibitor fadrozole and the steroidal inhibitor formestane (4-hydroxyandrostenedione). Fadrozole was superior to aminoglutethimide in terms of potency, selectivity, and safety [67], but its selectivity was not complete and clinical trials suggested that it was no more effective than tamoxifen [68, 69].
Fig. 2The development of aromatase inhibitors (AIs) has culminated in agents with high specificity and potency for aromatase. Spectrum of action of first- through third-generation AIs: The third-generation AIs act exclusively on the aromatase enzyme and do not appear to exert additional effects. Potency of AIs determined by degree of inhibition of total body aromatase: 4-OHA 4-hydroxyandrostenedione. Reprinted from [66] with permission from the Society of Endocrinology
To improve on fadrozole, Novartis synthesized a series of new compounds. Structure-activity relationship studies were then performed to identify the most potent AI from a series of benzyl-azole derivatives of fadrozole [70]. The third-generation AI letrozole (Femara®) was the result of this structure-activity approach to drug design and achieved the research goal of creating a highly potent and totally selective AI [71]. These compounds were also used to design pioneering molecular modeling techniques used to map the active site of aromatase [70, 72]. Other third-generation AIs developed during this period were the nonsteroidal agents vorozole (since discontinued) and anastrozole [73] (Fig. 2) [66] and the steroidal agent exemestane [74]. AIs have been classified as steroidal (type I; for example, exemestane) or nonsteroidal (type II; for example, letrozole and anastrozole) [75]. A comprehensive review of AIs focuses on the pharmacology and clinical development of letrozole [76].
Letrozole pharmacodynamics and pharmacokinetics
Potency
The chemical structure of letrozole (4,4′-[(1H-1,2,4-triazol-1-yl) methylene] bis-benzonitrile) is compared with other AIs in Fig. 3 [77]. The nitrogen-containing structures like the imidazoles and the triazoles bind to the iron in the heme moiety of CYP-450, whereas the cyanobenzyl moiety present in the nonsteroidal AIs such as letrozole partially mimics the steroid backbone of the enzyme’s natural substrate androstenedione. Furthermore, the triazole compound letrozole was found to be superior to other derivatives of fadrozole in terms of in vivo inhibition of aromatase [70].
Fig. 3Comparison of the molecular structures of aromatase inhibitors. Reprinted from [77] with permission from Elsevier
Letrozole is a highly potent inhibitor of aromatase in vitro, in vivo in animals, and in humans. The relative potencies of letrozole, anastrozole, and fadrozole were determined in a variety of model cellular endocrine and tumor systems containing aromatase (hamster ovarian tissue fragments, adipose tissue fibroblasts from normal human breast, the MCF-7Ca human breast cancer cell line transfected with the human aromatase gene, and the JEG-3 human choriocarcinoma cell line) [31]. These studies showed that although letrozole and anastrozole are approximately equipotent in a cell-free aromatase system (human placental microsomes), letrozole is 10–30 times more potent than anastrozole in inhibiting intracellular aromatase in intact rodent cells, normal human adipose fibroblasts, and human cancer cell lines (Fig. 4) [31]. In several other studies, letrozole has consistently demonstrated greater potency compared with anastrozole, exemestane, formestane, and aminoglutethimide (Table 1) [31, 71, 75, 78–82].
Fig. 4Relative potencies with which letrozole, anastrozole, and fadrozole inhibit aromatase from non-cellular and intracellular sources. Reprinted from [31] with permission from ElsevierTable 1Inhibitory concentrations of letrozole, anastrozole, exemestane, fadrozole, 4-hydroxyandrostenedione and aminoglutethimide against the aromatase enzyme derived from various cellular and non-cellular sources. Reprinted from [77] with permission from ElsevierAromatase inhibitorIC50 values (nM), (relative potency; letrozole = 1) Human placental microsomesParticulate fractions of human breast cancerRat ovarian microsomesMCF-7Ca cancer cellsJEG-3 cancer cellsCHO cellsHamster ovarian tissueHuman breastLetrozole2 (1)0.8 (1)Anastrozole8 (0.25)15 (0.053)Exemestane15 (0.13)5 (0.16)4-OHA30 (0.07)30 (0.027)AG20,000 (0.0001)10,000 (0.0008)Letrozole11 (1)0.07 (1)0.07 (1)20 (1)0.8 (1)Anastrozole23 (0.48)0.82 (0.085)0.99 (0.071)600 (0.033)14 (0.057)Fadrozole5 (2.2)0.05 (1.4)0.07 (1.0)30 (0.67)1 (0.80)4-OHA62 (0.18)AG1900 (0.0058)Letrozole1.02 (1)0.35 (1.0)0.45 (1)0.14 (1)Anastrozole5.35 (0.19)3.62 (0.097)5.66 (0.080)17.17 (0.0082)4-OHA0.59 (0.59)1.6 (0.28)0.72 (0.19)Letrozole7 (1)Anastrozole25 (0.28)Fadrozole7 (1)Letrozole1.4 (0)Anastrozole27 (0.052)4-OHA60 (0.023)AG5500 (0.00025)4-OHA 4-hydroxyandrostenedione, AG aminoglutethimideValues quoted are IC50 values representing the concentration needed to achieve 50% inhibition of aromatase activity. The relative potency of each inhibitor compared with letrozole is shown in parentheses
The degree of aromatase inhibition can be determined in vivo by measuring uterine weight after treatment with a standard dose of androstenedione in immature female rats [71]. Using this assay, it was found that the in vivo potency of letrozole is more than four orders of magnitude greater than aminoglutethimide (50% effective dose [ED50], 1–3 μg/kg vs. 30 mg/kg, respectively) [71]. It has also been shown that neoadjuvant letrozole profoundly inhibits in situ aromatase activity and reduces endogenous estrogens within the breast in postmenopausal women with large primary breast cancers [75].
In postmenopausal women, letrozole achieves significantly greater plasma estrogen suppression of estrogens and greater inhibition of in vivo aromatization than anastrozole [83]. In the study, levels of aromatase were detectable in 11 of 12 patients during treatment with anastrozole (mean percentage inhibition in the whole group, 97.3%) but in none of the 12 patients during treatment with letrozole (>99.1% suppression in all patients; Wilcoxon, P = 0.0022, comparing the two drug regimens). Suppression of estrone and estrone sulfate was found to be significantly greater during treatment with letrozole compared with anastrozole (P = 0.019 and 0.0037, respectively). Another study conducted in 54 postmenopausal women with invasive breast cancer showed that more complete inhibition of aromatase was achieved with 2.5 mg of letrozole than 1 mg of anastrozole, resulting in significantly greater suppression of estradiol (P < 0.0001), the most bioactive estrogen [84]. This recent study confirms previous observations showing that letrozole produces near complete inhibition of aromatase in peripheral tissues, associated with greater suppression of estrogen than achieved with other AIs [78, 85–90].
Selectivity
Letrozole is highly selective for aromatase and unlike first- and second-generation AIs does not significantly affect cortisol, aldosterone, or thyroxine [77]. In vitro studies showed that letrozole was more than three orders of magnitude more selective than aminoglutethimide in its effects on progesterone and corticosterone production, and more than 300-fold more selective against aldosterone than fadrozole [71, 78]. In vivo adrenocorticotrophic hormone (ACTH) stimulation tests in rats showed that letrozole had no significant effect on either aldosterone or corticosterone levels, even at a dose 1,000 times greater than that required for inhibition of aromatase [71].
The selectivity of letrozole has been demonstrated in clinical studies in postmenopausal women. These studies showed that letrozole has no effect on the plasma levels of 17α-OH progesterone, thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), or androstenedione and does not affect normal urine electrolyte excretion or thyroid function [86, 91–93]. Of note, the vast majority of patients treated with letrozole have a normal response to synthetic ACTH [86].
Anti-tumor activity in vivo
The potent anti-tumor effects of letrozole have been demonstrated in several animal models [77, 78, 94]. Letrozole induced complete regression of estrogen-dependent, 9,10-dimethylbenz-a-anthracene (DMBA)-induced mammary tumors in adult female rats [95]. The ED50 for letrozole was determined to be 10–30 μg/kg/day.
The use of MCF-7 cells transfected with human aromatase gene (MCF-7Ca) and implanted into athymic nude mice has proved to be an effective in vivo model for predicting clinical results with AIs [61, 96, 97]. Using this model, it has been shown that letrozole produces dose-dependent inhibition of tumor growth, resulting in complete inhibition at a daily dose of 10 μg/animal/day [94, 98]. Comparative studies using the MCF-7Ca model have shown that letrozole is more effective at suppressing tumor growth than the pure anti-estrogen fulvestrant and the SERM tamoxifen [99]. While anastrozole was also better than fulvestrant and tamoxifen in suppressing tumor growth, only letrozole was shown to induce tumor regression [99].
Another study, also using the MCF-7Ca model, demonstrated that letrozole potently inhibits mammary tumor growth but does not have the estrogenic effects of tamoxifen, as measured by its uterotrophic effects [100]. The observation that tamoxifen has an agonist effect even when estrogen synthesis is inhibited by letrozole suggests that there may be a degree of antagonism between these compounds [100]. Interestingly, studies in the MCF-7Ca model showed that letrozole is more effective as monotherapy than when combined with tamoxifen [80, 101]. In the study reported by Long et al. [101] tumor volume doubling times were 3–4 weeks in controls, 16 weeks with tamoxifen alone, 18 weeks with tamoxifen plus letrozole, and 34 weeks with letrozole alone. First-line treatment with letrozole was shown to be significantly superior to treatment with tamoxifen alone or with the two drugs combined (at week 16, both P < 0.001). Tumors that progressed during treatment with tamoxifen remained sensitive to second-line letrozole therapy, whereas tumors that progressed on letrozole did not respond to second-line treatment with tamoxifen or fulvestrant. In another series of experiments conducted by the same group using the MCF-7Ca model, letrozole was even effective as third-line therapy for a limited period when administered after treatment with tamoxifen and exemestane [102]. The studies showed that although exemestane was more effective than tamoxifen in controlling tumor growth, letrozole as first-line therapy was the most effective treatment overall, both in terms of the degree of tumor suppression and the length of effectiveness of treatment [102].
The potential of letrozole as a chemopreventive agent was investigated in an in vivo model using aromatase-transgenic female mice [103]. The model provided evidence to show that aromatase overexpression is sufficient to induce and maintain early preneoplastic and neoplastic changes that can be completely abrogated by treatment with letrozole. Carcinogenicity studies have also found that letrozole decreases the incidence of spontaneous mammary tumors and granular cell tumors in rats [104].
Pharmacokinetics of letrozole
Clinical pharmacokinetic studies of letrozole have been conducted in healthy volunteers [105–107] and in patients with breast cancer [108, 109]. Following oral administration, letrozole is rapidly and completely absorbed (mean absolute bioavailability of 99.9%) and extensively distributed to tissues. It has a large apparent volume of distribution at steady state (1.87 l/kg [range, 1.47–3.24]), and approximately 60% is bound to plasma proteins, mainly to albumin (55%). The terminal half-life (T1/2) of letrozole is 42 h. The terminal T1/2 was observed to be longer and area under the curve (AUC) greater in patients with breast cancer than in healthy volunteers, possibly due to reduction in metabolic clearance [109]. The major route of elimination of letrozole is metabolism by CYP-450 isoenzymes (CYP 3A4 and CYP 2A6) into an inactive carbinol metabolite. Systemic exposure to metabolites is, therefore, low. Steady-state concentrations of letrozole are reached after 2–6 weeks and maintained for long periods with no evidence of drug accumulation.
In marked contrast to the first-generation AI aminoglutethimide, no significant drug interactions have been reported for letrozole; however, when combined with tamoxifen, letrozole plasma concentrations are reduced by between 35% and 40% [110]. Age does not have an effect on the pharmacokinetics of letrozole. Exposure to letrozole, measured by AUC, is increased in renally impaired subjects but remains in the range seen in subjects without impaired function. However, hepatic impairment can markedly increase the T1/2 of letrozole, and caution is required in such patients.
Differences in pharmacokinetics, including uptake rates, elimination T1/2, and metabolism and clearance exist between AIs and have been reviewed by Lønning et al. [111]. The clinical significance of such differences is not known.
Clinical development of letrozole
Letrozole entered clinical trials on the basis of its high potency and selectivity for aromatase, the demonstration of unsurpassed anti-tumor effects in models of human breast cancer, and the development of a convenient oral formulation. Daily doses of 0.1–5 mg have been shown to suppress estradiol, estrone, and estrone sulfate plasma concentrations by 75–95% from baseline, while doses >0.5 mg suppress estrogens to below limit of detection [92, 112, 113]. Based on pharmacokinetic and pharmacodynamic studies, the recommended dose of letrozole is one 2.5 mg tablet once daily.
Preclinical models [97, 101] successfully predicted that letrozole would be superior to tamoxifen, the previous gold standard in the treatment of breast cancer. An extensive program of clinical trials has been conducted with letrozole across the spectrum of hormone-responsive breast cancer in postmenopausal women. The first randomized controlled trials demonstrated consistent superiority for letrozole compared with megestrol acetate, aminoglutethimide, and tamoxifen in patients with advanced breast cancer [114–118]. The clinical efficacy of letrozole in advanced breast cancer is described in a review by Dr. Mouridsen in this supplement.
Preclinical MCF-7Ca models have also predicted that letrozole should be clinically more effective than other less potent third-generation AIs [99, 102]. Letrozole (2.5 mg/day) and anastrozole (1 mg/day) were directly compared in a randomized, open-label phase IIIb/IV study involving 713 postmenopausal women with advanced breast cancer previously treated with an anti-estrogen [119]. While there was no difference between the treatment arms in the time to progression, letrozole produced a significantly higher overall response rate than anastrozole (19.1 vs. 12.3%, P = 0.013). Letrozole and anastrozole are currently being compared in a large randomized head-to-head trial in early breast cancer (ClinicalTrials.gov identifier NCT00248170) [120]. A review by O’Shaughnessy in this supplement provides the rationale for this trial and a description of its design.
The clinical benefits of letrozole in early breast cancer have already been demonstrated in landmark randomized clinical trials. MA.17 was the first trial to show improved clinical outcomes with extended adjuvant hormone therapy [121]. In this trial, letrozole given after initial adjuvant therapy with tamoxifen significantly improved disease-free survival compared with placebo [121, 122]. Full details of this trial are provided in a review by Dr. Goss in this supplement.
Subsequently, the Breast International Group 1-98 trial provided high-level evidence for the superiority of letrozole over tamoxifen as initial adjuvant therapy [123]. A detailed description of this ongoing trial, which will also help to define the optimal sequence for hormone therapies in hormone-responsive early breast cancer, is provided in a review by Dr. Thürlimann in this supplement. Letrozole has also demonstrated superior efficacy compared with tamoxifen when used as neoadjuvant therapy [124]. This treatment setting is particularly interesting in terms of drug development because the effects of hormone therapy on breast tumors can be detected early and may be predictive of long-term outcome [125].
Conclusions
Letrozole is a highly potent and selective AI that inhibits the enzyme activity of intracellular aromatase at the major sites where it is found, resulting in almost complete suppression of whole body aromatization. By effectively blocking estrogen synthesis, letrozole inhibits the growth or induces the regression of hormone-responsive breast tumors in vivo. Estrogen is implicated as a major risk factor in the majority of breast cancers; therefore, use of the most potent AI is a logical treatment strategy.
Studies conducted using in vitro and in vivo models have demonstrated that letrozole is the most potent of the third-generation AIs. Preclinical data obtained from MCF-7Ca xenograft models suggest that the greater potency of letrozole compared with anastrozole and exemestane may translate into clinically meaningful differences in postmenopausal women with hormone-responsive breast cancer. These models accurately predicted that letrozole would be more effective than tamoxifen in the clinical setting. The superiority of letrozole over tamoxifen has been consistently demonstrated in advanced and early breast cancer [118, 123]. Outstanding clinical questions, including what is the most effective AI and what is the optimal sequence for adjuvant hormonal therapy, will be answered by the results of ongoing trials involving letrozole.
In conclusion, experimental data indicating that letrozole efficiently inhibits aromatase activity have been confirmed clinically, leading to approved indications across the spectrum of breast cancer. The broad range of indications for letrozole in unique clinical settings is reshaping the management of hormone-sensitive breast cancer.

estrogen
breast cancer
aromatase
postmenopausal


BRCA1 and BRCA2 germline mutation analysis in the Indonesian population

Specific mutations in BRCA1 and BRCA2 genes have been identified in specific populations and ethnic groups. However, little is known about the contribution of BRCA1 and BRCA2 mutations to breast cancers in the Indonesian population. One hundred-twenty moderate to high risk breast cancer patients were tested using PCR-DGGE, and any aberrant band was sequenced. Multiplex ligation-dependent probe amplification (MLPA) was performed on all samples to detect large deletions in the two genes. Twenty-three different mutations were detected in 30 individuals, ten were deleterious mutations and 20 were “unclassified variants” with uncertain clinical consequences. Three of seven (c.2784_2875insT, p.Leu1415X and del exon 13–15) and two of four (p.Glu2183X and p.Gln2894X) deleterious mutations that were found in BRCA1 and BRCA2 respectively, are novel. Several novel, pathogenic BRCA1 and BRCA2 germline mutations are found in early onset Indonesian breast cancer patients, these may therefore be specific for the Indonesian population.
Introduction
Breast cancer is the most common cancer in women. In 5% to 10% of breast cancer cases, the disease results from a hereditary predisposition [1, 2], which can to a large extent be attributed to mutations in either of two tumour suppressor genes, BRCA1 (MIM# 113705) and BRCA2 (MIM# 600185) [3–5]. These genes are involved in DNA repair as well as transcriptional regulation [6, 7]. Women carrying pathogenic germline mutations in either of these genes tend to develop breast cancer at an early age [8, 9].
The BRCA1 and BRCA2 genes encode large proteins of 1,863 and 3,418 amino acids, respectively. Over 300 distinct mutations in BRCA1 and BRCA2 have been described [10, 11]. These mutations are widely scattered across both genes and most affect the structure and function of the gene. Nevertheless, a significant proportion (34% of BRCA1 and 38% of BRCA2 mutations) (http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic) are missense mutations that alter one amino acid, but do not truncate the protein and are rare sequence variants of unknown functional consequence. Moreover, a number of base substitutions do not alter the amino acid sequence or result in amino acid changes not associated with disease (polymorphisms) [12]. Hence the biggest challenge in interpreting the mutation analysis of BRCA1 and BRCA2 genes is to distinguish between harmless polymorphisms and deleterious mutations associated with increased cancer risk.
In addition, mutations specific for certain populations and ethnic groups have been identified in both genes. For example, specific BRCA1 and BRCA2 mutations were reported for Ashkenazi Jews [13]. Other common BRCA1 mutations were especially found in Italian, Canadian, Belgian or Dutch breast cancer families [14–16]. In Indonesia, the contribution of the BRCA1/BRCA2 mutations to the population incidence of early-onset breast cancer is largely unknown. In one pilot study, however, a new BRCA2 mutation was identified [17] indicating that it was worthwhile to more extensively study the Indonesian population, which was the aim of this study. The accumulating knowledge about the prevalence and nature of BRCA1 and BRCA2 mutations in specific populations may facilitate the interpretation of genetic analysis with regard to breast cancer risk of individual patients.
Materials and methods
Patients
A total of 120 unrelated breast cancer patients and 16 of their family members from three Indonesian cities (Jakarta and Jogjakarta on the Java island, Denpasar on the Bali island) were analyzed. Breast cancer patients at moderate to high-risk of a hereditary predisposition were selected according to the following criteria: (A) Breast cancer before the age of 41 (n = 102); (B) Two cases of breast cancer in the same family before the age of 60 (n = 9); (C) Three or more cases of breast cancer in the same family (n = 2); (D) Bilateral breast cancer (n = 7). Subjects were asked to fill out questionnaires to evaluate their personal and family histories, and blood specimens were collected for determination of BRCA mutations. Informed consent was obtained from all the subjects in this study.
DNA extraction and PCR amplification
Genomic DNA was isolated by the saturated salt extraction procedure as described in [18]. All 22 coding exons of BRCA1 and 26 coding exons of BRCA2 were amplified using primer sequences developed by the University of Groningen, The Netherlands [19]. Primers for DGGE were obtained from Ingeny (Goes, The Netherlands). Genomic DNA was amplified using 100–200 ng of template DNA, 10 pmol of the mixture of 40-mer primers, 30 mM of MgCl, 3 mM dNTPs (Invitrogen) and 0.7 unit of Platinum Taq (Invitrogen) in 9 μl PCR reactions. In order to speed up the test, the PCR reaction was placed in 384 well plates using a pipetting robot (TECAN Miniprep 75). PCR conditions were performed as previously described [17].
Denaturing Gradient Gel Electrophoresis and DNA sequencing
A 4–6 μl aliquot of each PCR product with relatively large melting temperature differences were pooled as previously described [17] with some modifications for optimal results. The fragment pool was designed based on melting profiles and sequence. Electrophoresis was performed in 0.5 TAE buffer at 58°C, 120 V for 16 h for BRCA1 gene, and 55°C, 100 V for 18 h for BRCA2. Gels were stained with ethidium bromide and photographed under a UV transilluminator. The aberrantly migrating samples were re-amplified using sequencing primers and sequencing was performed using Big Dye Cycle-sequencing kit according to the manufacturer’s instructions. The reaction products were analyzed using an ABI 3100 DNA Sequencer (Applied Biosystems, Torrence, CA, USA) and sequence files were edited using the Bio Edit program. The classification of gene alterations was performed in accordance with the entries in the Breast Cancer Information Core (BIC, Bethesda, MD).
Multiplex ligation-dependent probe amplification (MLPA)
The principle of the MLPA technique has been described elsewhere [20]. The MLPA test for BRCA1 (P02) and BRCA2 (P45) mutations were obtained from MRC-Holland, Amsterdam, The Netherlands. The fragments were analyzed on an ABI model 310 capillary sequencer (Applied Biosystems, Torrence, CA, USA) using Genescan-TAMRA 500 size standards (Applied Biosystems). Fragment analysis was performed with Genescan software.
Results and discussions
We identified 120 incident Indonesian breast cancer cases diagnosed before the age of 41 years, or having family history of breast cancer, or harboring bilateral breast cancer during September 1999–April 2005 (Jogjakarta) and during July 2004–April 2005 (Jakarta and Denpasar). In addition, 16 of their family members were analyzed.
The entire coding regions and exon-intron junctions of BRCA1 and BRCA2 were screened in these 136 persons of breast cancer patients and their families using PCR-DGGE (Fig. 1) followed by sequencing (Fig. 2) for samples with aberrant migrating bands. To optimize the screening, MLPA, a relatively new technique, was also performed in all samples (Fig. 3). Here, we report on 116/120 women (96.7%) for whom BRCA1/2 analysis were completed. The remaining four patients (all from group A) had to be excluded due to the small amount of extracted DNA that did not allow complete screening of the BRCA1 and BRCA2 genes.
Fig. 1DGGE analysis of fragments 11.15 g, 11.4 and 11.10 of the BRCA2 gene in ten unrelated breast cancer patients. The arrows show altered band mobility compare to other patientsFig. 2Sequence electropherogram of a normal individual showing (A) wild-type BRCA2 exon 11 sequence and (B) of breast cancer patient (B-3-5) showing c.2699_2704delTAAATG mutationFig. 3MLPA analysis of BRCA1 gene of patient sample (blue) compare to the normal control (red). X and Y axis represent peak size and peak height respectively. There are reduced peaks in the patient sample compared to the normal control in exons 13, 14 and 15 (arrows) indicating deletions
BRCA1 and BRCA2 pathogenic mutations
The analysis of 116 unrelated breast cancer patients with breast cancer revealed that nine patients (7.8%) carried pathogenic germline mutations especially the early onset patients: 3 within BRCA1 (2.6%) and 6 within BRCA2 (5.2%) which is comparable to previous studies [21]. We only found BRCA1 and BRCA2 mutations in groups A (“early onset”, n = 7 out of 98, 7.1%) and B (two cases of breast cancer in the same family before the age of 60, n = 2 out of 9 (22.2%)) (Table 1). There were twice as many BRCA2 mutations as BRCA1 mutations. Although the absolute numbers are low and no firm conclusions can therefore be drawn, this is comparable to other Asian regions [22–24] but seems to discern the Indonesian population from non-Asian ethnic groups where the reverse trend is seen.
Table 1BRCA1 or BRCA2 germline mutations in Indonesian women with early onset breast cancerPatientAgeageneExonMutationbmutation typePathogenic mutationBICcAE25BRCA111c.2784_2785insTframeshift+noB1031BRCA113p.Leu1415Xnonsense+noAA40BRCA113–15−dlarge rearrangement+noAB34BRCA211c.3040_3043del4frameshift+1B566BRCA211p.Glu2183Xnonsense+noB665BRCA211p.Glu2183Xnonsense+noB-III-530BRCA211p.Leu824Xnonsense+noAZ40BRCA211p.Leu824Xnonsense+noW-II37BRCA221p.Gln2894Xnonsense+noQ-II40BRCA12c.101–10T>CIVS±6P-III-1919BRCA19p.Val191IleMissense±6J2232BRCA111p.Leu1209ValMissense?noAZ40BRCA116p.Met1652IleMissense±35B1 24BRCA120c.5313–31A>GIVS?noB731BRCA124p.Arg1835GlnMissense?no21633BRCA124p.Thr1852IleMissense?noP-III-1919BRCA25p.Gln147ArgMissense±6B324BRCA210p.Gln609GluMissense?noC-II-739BRCA211p.Met1149ValMissense±5AO28BRCA211p.Met1149ValMissense±5AQ44BRCA211p.Met1149ValMissense±5BH38BRCA211p.Met1149ValMissense±517236BRCA211p.Gln699LeuMissense?noJ3229BRCA211p.Arg2108CysMissense±16J633BRCA211p.Val950IleMissense?no20637BRCA225c.9485–16T>CIVS±4BC35BRCA227p.Ile3412ValMissense±10916633BRCA227p.Ile3412ValMissense±109J2435BRCA227p.Ile3412ValMissense±10920637BRCA227p.Lys3326Xnonsense ±289aAge at time of diagnosisbGen Bank Accession number, BRCA1: U14680, BRCA2: U43746cnumber of times reported in BICdnot determined, detected by MLPA
Seven pathogenic mutations were found in nine probands: three in BRCA1 (c.2784_2785insT, pL1415X (c.4361_4362insT), del exon 13–15) and four in BRCA2 (c.3040_3043delGCAA, p.Glu2183X (c.6775G>T), p.Leu824X (c.2699_2704delTAAATG), p.Gln2894X (c.9008C>T)). All these mutations were classified as pathogenic as they are predicted to result in protein truncation. The three pathogenic mutations found in BRCA1 were not previously reported in the BIC database as well as two novel nonsense mutations (p.Glu2183X and p.Gln2894X) identified in BRCA2. The p.Glu2183X mutation was found in 2 related patients that had breast cancer above the age of 60.
One of seven pathogenic mutations found in BRCA1 and BRCA2 showed a significant clinical impact on the patient (Table 2). Patient AE with a one nucleotide insertion (Thymine) between nucleotide 2784 and 2785 (c.2784_2785insT) in exon 11 of BRCA1 suffered from bilateral breast cancer at a relatively early age (25 years). The insertion leads to frameshift and creates a premature stop codon in exon 11. The mutation takes place in the sequence within BRCA1 encoding for aminoacids 758–1064 which interact with RAD51 protein that is required for homologous recombination (HR) repair of double strand breaks (DSBs) [25], which is one of the most important functions of the BRCA1 protein. This patient presented in a late stage (stage III for both breasts) and only survived for 9 weeks after treatment. Her mother did not carry this mutation. Although her father may be carrier, the mutation is probably de novo as there was no family history of breast or other cancers.
Table 2Clinicopathological features of Indonesian breast cancer patients with deleterious BRCA1 or BRCA2 germline mutationsPatientAgeaGene with germline mutationMutationbstageDiagnosisMenopausal statusfamily history of cancerSurvival statusAE25BRCA1c.2784_2785insTIIIB/IIIAIDC, bilateralpreNoDOD 9 wB1031BRCA1p.Leu1415XIIDCpreNoDOD 57 wAA40BRCA1-cIIIBIDC N+preNoAWDAB34BRCA2c.3040_3043del4IIIBIDC N+preSister, IntDOD 17 wB563BRCA2p.Glu2183XIVTubularpostSister,BrAWDB665BRCA2p.Glu2183XIIIIDCpostBrother, BrAWDB-III-530BRCA2p.Leu824XIIDCpreNoAWDAZ40BRCA2p.Leu824XIVIDCpreSister, CvDOD 46 wW-II37BRCA2p.Gln2894XIIIAIDCpreNoDOD 107 waAge at time of diagnosisbGen Bank Accession number, BRCA1: U14680, BRCA2: U43746cnot determined, detected by MLPAIDC: invasive ductal carcinoma; DOD: dead of disease; bil: bilateral breast cancer; N+: with metastatic to lymph node; Int: intestinum cancer, Br: breast cancer; Cv: cervical cancer
The second pathogenic mutation with a significant clinical manifestation was a cytosine for thymine substitution on nucleotide 9008 of BRCA2 leading to a premature stop codon in position 2894, c9008C>T (p.Gln2894X). Patient W presented at age 37 in a late stage and survived for only 107 weeks after initial treatment. She had no family history of breast or other cancers. This mutation lies within exon 21 of BRCA2 which is the proposed site for interaction with the DSS1 protein that seems to have a fundamental role in enabling the BRCA2-RAD51 complex to associate with sites of DNA damage [26].
The c.2699_2704delTAAATG (p.Leu824X) in BRCA2 that has been reported previously by us in the Indonesian population [17], was found in one other patient in the present study (Table 1). This mutation lies in exon 11 BRCA2, within the BRC repeats domain. The truncating mutation causes loss of three quarters of the protein leading to lack of interaction with the RAD51 protein. Different from BRCA1, the repair of DSBs by HR is the most important function of the BRCA2 protein [27]. Patient B-III-5 was diagnosed with early stage breast cancer at age 30 with no family history of breast or other cancers. Her sister carried the same mutation, but with no present clinical manifestation as yet. Patient AZ who was diagnosed at 40 years of age, presented in late stage, only survived 46 weeks after initial treatment. This patient also harbored a mutation in exon 16 of BRCA1, a G to A substitution in nucleotide 5075 (c.5075G>A), which leads to amino acid change from Methionine to Isoleucine, (p.Met16521Ile) which has to date been reported 35 times in BIC as a UV mutation. As the c.2699_2704delTAAATG mutation was found in two unrelated patients, this mutation could be a good candidate as a founder mutation.
None of the families with more than 3 cases of breast cancer and families with bilateral breast cancer showed pathogenic mutations in the BRCA1 and BRCA2 genes. Family U had four first-degree relatives that were affected by breast cancer. Two of four members had bilateral breast cancer. In spite of this high familial breast cancer incidence, no BRCA1/2 mutations were found.
BRCA1 and BRCA2 unclassified variants
Sixteen (7 BRCA1 and 9 BRCA2) rare mutations of so far unknown significance (“unclassified variants”, UVs) were detected in 18 patients: 13 missense changes and 3 intronic variants. Of these 16 UVs, 7 were novel, whereas the other UVs have been previously reported in the BIC database (Table 1). From the 18 patients which carried UV mutations, two patients were detected in families from group D; one patient in a group B family and the other fifteen patients in families from group A.
Seven UV were found in the BRCA1 gene, two mutations occurring in the intronic region between exons 1 and 2 (c.101–10T>C) and between exons 19 and 20 (c.5313–31A>G), and five missense mutations identified: p.Val191Ile (c.690G>A), p.Leu1209Val (c.3744T>G), p.Met1652Ile (c.5075G>A), p.Arg1835Gln (c.5623G>A) and p.Thr1852Ile (c.5674C>T).
Four out of seven BRCA1 missense mutations; p.Leu1209Val (c.3744T>G), c.5313–31A>G, p.Arg1835Gln (c.5623G>A) and p.Thr1852Ile (c.5674C>T) were have not been described previously in the BIC. The p.Leu1209Val may not be a significant change as both Leucine and Valine belong to the same group of non polar amino acids. However, p.Arg1835Gln is possibly an important alteration since a positively charged Arginine is replaced by an uncharged Glutamine, which may have an effect on the structure and/or function of the protein. Another potentially important alteration concerns p.Thr1852Ile, where the hydrophilic amino acid Threonine is replaced by a hydrophobic Isoleucine. The sites of mutation of both p.Arg1835Gln and p.Thr1852Ile also have to be considered as they lie within the site for the activation domain of the BRCA1 protein [28]. The intronic UV c.5313–31A>G also deserves further investigation as it may theoretically have an effect on splicing. However, according to splice site finder (http://www.genet.sickkids.on.ca/∼ali/splicesitefinder.html), the splicing sites in the wild type and mutant alleles are similar, so therefore we can suggest that the c.5313–31A>G has no effect on splicing.
Nine different UVs of the BRCA2 gene were found in fourteen patients (Table1), and three of them were novel; p.Gln609Glu 9c.2053C>G), p.Gln699Leu (2324A>T) and p.Val950Ile (3076G>A). One truncating mutation near the C-terminal end of BRCA2, p.Lys3326X (c.10204A>T) is probably not pathogenic. Since the truncating mutation is at the very end of the protein, it is possible that protein functions are not affected. Most of the few entries in databanks describing nonsense mutations near the C terminus of BRCA2 between codon 3308 and 3408 are described as UVs. Thus, the effect of this truncating mutation on cancer predisposition remains unclear.
The p.Val950Ile may not be a significant change as both Valine and Isoleucine belong to the same group of non polar, hydrophobic amino acids. However, p Gln609Glu and p.Gln699Leu are potentially important alterations as for p.Gln609Glu, a non acidic, polar, hydrophilic Glutamine is replaced by a negatively charged Glutamic acid, whereas for p.Gln699Leu, an uncharged hydrophilic Glutamine is replaced by a hydrophobic Leucine. As it takes place within the BRC repeats of the BRCA2 protein, the p Gln699Leu alteration might affect protein structure and function.
To know more about the importance of amino acid substitutions for protein function, we compared the amino acid sequence of interest in seven other species, i.e. Mus musculus, Rattus rattus, Bos taurus, Gallus gallus, Canis familiaris, Macaca mullata and Monodelphis domestica. The missense mutation p.Leu1209Val lies in the conserved region of exon 11 of the BRCA1 gene as the sequence is maintained in seven other species, whereas p.Arg1835Gln and p.Thr1852Ile are only conserved in four and three other species (comparison of p.Arg1835Gln and p.Thr1852Ile with Bos taurus sequence is not possible because the BRCA1 gene is shorter). Therefore, even tough the Leucine to Valine changes may not give any effect on amino acid charge, its conservation in evolution is suggestive of a functional role. Interestingly, p.Gln609Glu and p.Gln699Leu of BRCA2 that result in a quite dramatic amino acid subtitution that might lead to protein structure changes, are only conserved in four and five species respectively. As for the p.Val950Ile, the conservation in evolution is quite low. Although p.Gln609Glu is less conserved, we still believe that Glutamine to Glutamic acid substitution may have an effect on protein conformation as two adjacent acidic amino acids will be formed as the result of the substitution.
Glycosylation moiety of an amino acid also plays a role in protein function. Amino acid substitutions involving Serine, Threonine and Asparagine, should also be checked for their O-GlcNac potential and threshold. Here we have a Threonine to Isoleucine substitution (p.Thr1852Ile) that after checking with YinOYang (http://www.cbs.dtu.dk/services/YinOYang) showed no significant threshold changes between the wildtype and the mutant allele.
The possible effect of amino acid changes in proteins can also be assessed using similarity scores (based on Grantham table [29]), in which a value above 100 for an amino acid substitution indicates a higher chance of impact on protein function. Among seven novel UVs in the BRCA1 and BRCA2 genes found in the present study, only p.Gln699Leu in BRCA2 has a similarity score above 100, whereas p.Gln609Glu and p.Val905Ile in BRCA2 have the lowest score (Table 3).
Table 3The amino acid properties of novel unclassified mutations in BRCA1 and BRCA2 within an Indonesian breast cancer populationGeneAmino acid changeChange of chargeChange of amino acid groupSimilarity scorea# species with conserved sequenceBRCA1Leu to ValNoneNo327a,b,c,d,e,f,gBRCA1Arg to GlnPos to no chargeYes434a,c,f,gBRCA1Thr to Ilepolar to non polarYes893a,c,gBRCA2Gln to GluNo charge to negYes294a,b,c,gBRCA2Gln to LeuPolar to non polarYes1135a,b,d,e,fBRCA2Val to IleNoneNo292f,gabased on Grantham table [Grantham et al. [29], a score above 100 indicates significance changesa = Macaca mullata, b = Bos taurus, c = Canis familiaris, d = Rattus rattus, e = Mus musculus, f = Gallus gallus, h = Monodelphis domestica
Overall, we propose that among the seven novel UVs, there are three mutations that are possibly pathogenic: p.Leu1209Val for its location in a conserved region, and p.Gln609Glu and the p.Gln699Leu because of two adjacent acidic amino acid being formed and a high similarity score, respectively.
When comparing the three different Indonesian regions, the percentages of breast cancer patients with pathogenic BRCA1/2 mutations was significantly higher in Denpasar (Bali island) than in Jogjakarta and Jakarta (Java island) ((25% (3/12), 7.2% (6/83) and 0% (0/25) respectively (P = 0.0255, chi-square test)). The percentages of breast cancer patients with UV mutations in Jakarta, Jogjakarta, and Denpasar were 16% (4/25), 12% (10/83), and 25% (3/12), respectively (n.s.). Although the number of patients is too small to draw firm conclusions, these data may point to geographic differences within Indonesia.
It was initially suggested that the BRCA1 and BRCA2 genes would be responsible for most cases of inherited breast cancer, but more recent studies suggest that they would account for a far smaller proportion, with considerable variation among different populations [30]. We found that the incidence of mutations in these genes varies, depending on the diagnostic group. In this sense, mutations were present in (22/102) 21.6% of early onset patients (group A), 28.7% (2/7) in patients with bilateral breast cancer (group D) and (2/9) 22.2% of patients with two cases of breast cancer before the age of 60 (group B). The proportion of families affected by BRCA1/2 mutations depends on the population analyzed and on the criteria used to select the patients. Family history of breast cancer was, however, absent or not suggestive of a hereditary predisposition in three-fourth of the deleterious mutations carriers and in more than 90% of UV carriers. This suggests that BRCA screening policies based on family history only would miss a considerable proportion of mutation carriers.
In conclusion, a relatively high percentage of early onset Indonesian breast cancer patients carry a germline mutation in either BRCA1 or BRCA2. Several novel, pathogenic BRCA1 and BRCA2 germline mutations have been found, as well as a variety of novel “unclassified variant” mutations that may therefore be specific for the Indonesian population. It is likely that some of the “unclassified variant” mutations may have a functional role in breast cancer development, which deserves to be explored further.


Remyelination after chronic spinal cord injury is associated with proliferation of endogenous adult progenitor cells after systemic administration of guanosine

Axonal demyelination is a consistent pathological sequel to chronic brain and spinal cord injuries and disorders that slows or disrupts impulse conduction, causing further functional loss. Since oligodendroglial progenitors are present in the demyelinated areas, failure of remyelination may be due to lack of sufficient proliferation and differentiation of oligodendroglial progenitors. Guanosine stimulates proliferation and differentiation of many types of cells in vitro and exerts neuroprotective effects in the central nervous system (CNS). Five weeks after chronic traumatic spinal cord injury (SCI), when there is no ongoing recovery of function, intraperitoneal administration of guanosine daily for 2 weeks enhanced functional improvement correlated with the increase in myelination in the injured cord. Emphasis was placed on analysis of oligodendrocytes and NG2-positive (NG2+) cells, an endogenous cell population that may be involved in oligodendrocyte replacement. There was an increase in cell proliferation (measured by bromodeoxyuridine staining) that was attributable to an intensification in progenitor cells (NG2+ cells) associated with an increase in mature oligodendrocytes (determined by Rip+ staining). The numbers of astroglia increased at all test times after administration of guanosine whereas microglia only increased in the later stages (14 days). Injected guanosine and its breakdown product guanine accumulated in the spinal cords; there was more guanine than guanosine detected. We conclude that functional improvement and remyelination after systemic administration of guanosine is due to the effect of guanosine/guanine on the proliferation of adult progenitor cells and their maturation into myelin-forming cells. This raises the possibility that administration of guanosine may be useful in the treatment of spinal cord injury or demyelinating diseases such as multiple sclerosis where quiescent oligodendroglial progenitors exist in demyelinated plaques.
Introduction
Demyelination contributes to functional deficits in central nervous system (CNS) disorders [1–3]. Traumatic spinal cord injury (SCI) induces local inflammation and demyelination in the white matter around the lesion resulting in disrupted axonal conduction [4–7]. Remyelination is critical for recovery, but does not occur spontaneously [8–11]. Spinal cords contain endogenous progenitors that can proliferate and differentiate into mature oligodendroglia and remyelinate axons under certain circumstances [12–14]. After acute SCI in rats, oligodendrocyte precursors proliferate in the first 2 weeks but are “silent” thereafter [15], possibly because the oligodendroglial precursors cannot differentiate into mature oligodendroglia capable of remyelinating axons. Enhancement of proliferation of oligodendrocytes or progenitors may lead to repair and restoration of function after SCI.
Neurotrophic factors restore physiological function by promoting oligodendrocyte proliferation and axonal sprouting [16–19], regulating the proliferation of oligodendrocyte progenitors [3] and modulating the differentiation [20–22] and the maturation of oligodendrocyte precursors into myelin-forming cells [23, 24].
Guanosine has trophic effects on many cell types [25–28], and we have some evidence that guanine acts similarly. For example, it stimulates proliferation of a variety of cells in culture [25, 28] and promotes synthesis and release of several potentially neuroprotective trophic factors from a variety of cells, including neuron growth factor (NGF) from astrocytes as well as basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF beta) [25, 27]. Extracellular guanosine itself also enhances outgrowth of nerve processes from PC12 cells and from primary cultures of embryonic rat brain neurons [25]. As well, it acts synergistically with NGF to promote outgrowth of neurites [29, 30].
Our preliminary data [31] showed that 35 days after moderate traumatic injury of the spinal cord of rats, when the initial spontaneous incomplete recovery of function had plateaued, systemic administration of guanosine (intraperitoneally 8 mg/kg body weight) for 7 days enhanced locomotor functional recovery that continued to improve throughout the 7-day duration of the treatment and correlated with the increase in myelination. In the present study, we treated rats for up to 14 days to determine whether guanosine-induced functional improvement was due to triggering the endogenous adult progenitors. Meanwhile, we also examined the uptake and tissue distribution of systemically administered guanosine.
Materials and methods
All experiments were performed in compliance with the requirements of the Animals for Research Act of Ontario, Canada and the Guidelines of the Canadian Council on Animal Care, and had been approved by the Animal Research Ethics Board of McMaster University.
Spinal cord injury Adult female Wistar rats (280–300 g weight, Charles River) were anaesthetized with isoflurane (3–5%): O2 (1 l/min). Buprenorphine (0.03 mg/kg body weight, subcutaneously) was administered prior to surgery for pain relief. Moderate spinal cord injury was induced by crushing exposed cords with modified coverslip forceps [31–33]. The forceps were closed slowly over 2 s and the compression sustained for 15 s. The muscles and fat pad over the lesion were sutured and the skin was closed with stainless steel clips. Post-operatively, the rats were kept quiet and warm [31, 34, 35].
Behavioural assessment and drug administration Rats were handled daily for 2 weeks pre-operatively to acclimatize them to the experimental procedures and behavioural test. After the spinal injury, locomotor recovery was assessed weekly for 35 days in an open field walking task (OFWT) before treatment. Cagemates (two animals) were placed in the centre of the open field, formed by a child’s circular plastic swimming pool (1.3 m in diameter). They were observed for 5-min periods and scored for general locomotor ability using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale [36, 37]. Rats were rated on a scale of 0 to 21, with 0 being no function and 21 being normal. If the animal stopped moving for a minute, it was placed again in the centre of the open field; otherwise it was left alone for the duration of the 5-min test period.On day 35 following the injury, rats were randomly assigned to three groups. One group of rats was used for baseline behavioural and histological analysis. The other rats received either daily intraperitoneal (i.p.) injections of 8 mg/kg guanosine or the same volume of saline containing 0.001 N NaOH [31] for 2 weeks. The open field walking testing was performed once a day from days 35 to 48 post-surgery (0 to 14 days post-treatment). Groups of 9–12 rats were killed by perfusion/fixation for immunohistochemical analysis at 1, 3, 7 or 14 days after treatment.
BrdU administration Bromodeoxyuridine (BrdU) was used to label proliferating cells after treatment. BrdU (50 mg/kg in sterile saline; Sigma, B-5002) was injected intraperitoneally daily for 1, 3, 7 or 14 consecutive days, beginning the day treatment was initiated, thereby reflecting cell proliferation over the entire treatment period.
Tissue processing and immunohistochemistry On post-operative days 35, 36, 38, 42 or 49 (representing baseline, 1, 3, 7 or 14 days treatment), rats were deeply anaesthetized with sodium pentobarbital (50–60 mg/kg body weight, i.p.) immediately following the final locomotor testing and perfused transcardially with 100 ml 0.1 M phosphate-buffered saline (PBS) containing 0.1% heparin, followed by 300–500 ml of 4% paraformaldehyde (PFA). T9 to L1 segments of the spinal cords were removed and incubated in the same fixative for 2 h at room temperature and then cryoprotected in 30% sucrose PBS solution. A segment of each cord, extending from 5 mm rostral to 5 mm caudal to the lesion site was embedded in medium (Tissue-Tek® O.C.T. compound, Sakura Finetek USA, Inc., Torrance, CA, USA). Serial transverse sections were cut at 15- to 20-μm intervals on a cryostat and mounted onto slides (ColorFrost/Plus; Fisher, Pittsburgh, PA, USA). Every fifth section was stained with the lipophilic dye Luxol fast blue to stain myelin [38]. Adjacent sections were stained immunohistochemically with the various antibodies described below.For BrdU immunohistochemistry, 6% hydrogen peroxide was used to reduce endogenous peroxidase activity, followed by three 10-min PBS rinses and incubation in 1 N HCl at 37°C for 30 min. After rinsing, a blocking solution [5% bovine serum albumin (BSA) in 0.1% Triton-100] was applied for 1 h; sections were subsequently incubated in rat anti-BrdU (1:200 in 1% BSA in 0.1% Triton-100; Serotec, Raleigh, NC, USA) at 4°C overnight in a humid chamber. Sections were then rinsed and incubated with biotinylated horse anti-rat IgG secondary antibodies. Sections were then incubated with Elite ABC (Vector Laboratories, Burlingame, CA, USA) for 1 h, followed by 3,3′-diaminobenzidine (DAB) substrate kit for peroxidase (Vector Laboratories, Burlingame, CA, USA) for 5–10 min. Sections were dehydrated, coverslipped and examined under a microscope.To determine the fate of newly born cells after SCI, double-fluorescent immunolabelling was performed, combining BrdU with one of the cell-specific phenotypic markers listed below. For double immunofluorescence, sections were pretreated with HCl as described above and incubated with BrdU antibody mixed with one of the following antibodies in PBS at 4°C overnight in a humid chamber: rabbit anti-MBP (myelin basic protein) polyclonal antibodies (1:50; Chemicon Int., Temecula, CA, USA), rabbit anti-NG2 polyclonal antibodies (chondroitin sulfate proteoglycan for oligodendroglial progenitors; 1:200; Chemicon Int. Temecula, CA, USA), mouse anti-Rip monoclonal antibodies (mature oligodendrocyte-specific marker: Reactive Immunology Protein; 1:200; Chemicon Int., Temecula, CA, USA; [39]), rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibodies (1:600; Zymed® Lab-SA System Kit, San Francisco, CA, USA) and mouse anti-microglial-specific marker OX-42 monoclonal antibodies (1:200; CD11b, Research Diagnostics Inc., Flanders, NJ, USA). For Rip and OX-42 monoclonal antibodies single staining, sections were developed with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. For BrdU and polyclonal antibodies (NG2 or MBP or GFAP) double immunolabelling, sections were developed using a mixture of FITC-conjugated goat anti-rat IgG and rhodamine-conjugated goat anti-rabbit IgG in 1% normal goat serum and 0.25% Triton X-100; 1:200 (Invitrogen, Carlsbad, CA, USA) for 2 h. To examine the contribution of Schwann cells from the peripheral nervous system (PNS) in the remyelination process, sections were immunolabelled with specific markers for Schwann cell myelin P0 (a rabbit polyclonal anti-P0 antibody was kindly donated by Prof. Marie T. Filbin, Department of Biology, CUNY, NY, USA; 1:200) and secondary antibody using rhodamine-conjugated goat anti-rabbit IgG. For detection of apoptotic cells, a terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labelling (TUNEL) stain was performed using the ‘In situ Cell Death Detection Kit, Fluorescein’ (Roche Molecular Biochemicals, Mannheim, Germany), according to the manufacturer’s instructions.
Quantification To quantify the amount of myelin present at the injury site before and after treatment, the Luxol fast blue-stained section from each cord (n = 7–10 for each group) that contained the greatest lesion area was selected together with the adjacent two caudal and two rostral Luxol blue-stained sections. Luxol fast blue staining was also done on cords of five additional unoperated normal animals for comparative purposes. Digital photographs were taken of the sections. The total area of each section and the area of the fast blue-stained portion were measured using a computerised Bioquant BQ-TCW98 image analysis program by an investigator who was blind to group assignment.For quantification of cell proliferation, five to seven sections taken from the penumbra of the lesion and spaced about 100 μm apart were analysed for each animal (n = 7–9 animals per group). Cellular proliferation was determined by counting the total number of BrdU+ nuclei in spinal cord cross sections. Low power sections were digitized and manually outlined using an image analysis system. Any cavities present in the sections were excluded from analysis. Positively labelled nuclei were counted automatically. The threshold was set such that only positively labelled profiles of the appropriate size were counted. Data are expressed as cells per section.The total number of NG2+ progenitor cells and the number of those that proliferated (double-labelled for NG2 and BrdU: NG2+/BrdU+) was determined by manual counting. Cell counting was done conservatively, and a positive profile was counted as a cell only when clearly defined borders could be detected completely around and adjacent to the entire nucleus. Data are expressed as the number of immunostained cells per section.Mature oligodendrocytes in injured cords were identified with Rip, a specific marker for mature oligodendrocytes that recognises an unknown epitope [39] and has been used successfully in the CNS to label both oligodendrocyte processes and myelin sheaths. The number of mature oligodendrocyte (Rip+ cells) in the tissue sections was determined by manual counting.
Evaluation of exogenous guanosine distribution A guanosine solution (8 mg/kg) was administered intraperitoneally to rats with spinal cord injury induced 5 weeks previously. Samples of spinal cord were taken from the site of the lesion and immediately above or below the lesion just before the intraperitoneal injection of guanosine and at 7.5, 30 and 60 min after the guanosine injection. There were between three and six animals at each time point. Samples of spinal cord were placed in 0.5 ml of cold 0.4 M perchloric acid solution and immediately homogenized. Samples were then centrifuged (4°C, 10,000 g, 10 min) to remove proteins precipitated by perchloric acid and the supernates containing the soluble molecules were neutralized with KOH solution and centrifuged (4°C, 10,000 g, 5 min). Samples were filtered with 0.2-μm filters (Millipore, Billerica, MA, USA) and stored at −70°C before high-performance liquid chromatography (HPLC) [40] or capillary electrophoresis (CE) analysis.
Tissue concentrations of guanosine and guanine To evaluate the concentration of guanosine and its principal degradation product, guanine, in spinal cords before and after the intraperitoneal injection of guanosine, either HPLC or CE was used. There was no significant difference in the values obtained by these two methods so the data represent the mean of values obtained by both analyses. HPLC analysis was carried out using an Agilent 1100 series HPLC (Agilent Technologies, Waldbronn, Germany) and an ion-pair technique. Separation was carried out with a reverse-phase analytical column (LiChroCART 125-4 LiChrospher 100 RP-18 5 μm, Merck, Darmstadt, Germany) and the flow rate was 1.5 ml/min. A 15-min linear gradient was applied from 100% buffer A (60 mM KH2PO4 and 5 mM tetrabutylammonium phosphate, pH 6) to 100% buffer B (30% methanol plus 70% buffer A). CE separations were carried out using an HP3DCE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detection system. Analysis was performed using a 80.5 cm × 50 μm ID × 375 μm OD uncoated fused silica capillary from Agilent Technologies (Waldbronn, Germany) with an effective length of 72 cm to the detector window. The capillary temperature was kept constant at 37°C and the running buffer was 10 mM borate, pH 9.3. The samples were injected by pressure (50 mb for 40 s) and the separation was conducted at 22 kV. Detection took place at 254 nm and purines were identified by their migration times and UV spectra using the diode array detector (Agilent Technologies, Waldbronn, Germany).
Statistical analysis Data are expressed as mean ± SEM. Behavioural scores were analysed by the Kruskal-Wallis non-parametric analysis of variance (ANOVA). Post hoc comparisons were made using the Dunnett’s test. Correlation between behavioural and histological outcomes was analysed using regression analysis. Significance was set at p < 0.05.
Results and discussion
After SCI, motor function in the legs partially recovered over 3–4 weeks, after which there was no further improvement (Fig. 1a). Daily guanosine for 14 days (8 mg/kg intraperitoneally beginning 35 days after injury) produced improvement in locomotor performance over the next 2 weeks; control rats showed no improvement (Fig. 1b). This treatment regimen was associated with significantly increased amounts of myelin in the penumbra of the cord lesions shown by Luxol fast blue staining (Fig. 1c–e). The extent of Luxol fast blue staining in the injured cords correlated with each animal’s open field walking scores (r = 0.72; p < 0.05; Fig. 1f), indicating that guanosine-induced functional recovery may be related to the increase in myelin. Immunostaining for CNS myelin basic protein (MBP; Fig. 1g,h) and a specific marker for Schwann cell myelin, P0 (Fig. 1i,j) showed that Schwann cells from the peripheral nervous system did not enter spinal cord and, therefore, did not contribute to the remyelination.
Fig. 1Systemic treatment with guanosine improves locomotor function and correlates with increased myelination in rats with stable chronic spinal cord injury. a After spinal cord crush, locomotor function was assessed in an open field walking task (OFWT) and scored using a standard scale (“Materials and methods”). No further improvement in locomotor performance occurred after 28 days. b On days 35–48 after injury (days 1 to 14 of treatment in the figure; the first test done immediately prior to the first treatment), animals were randomly divided into two groups. One group received guanosine (▴), the other group received vehicle (♦). The locomotor function in the control group did not change significantly throughout the 14 days of vehicle administration. In contrast, rats that received guanosine showed a marked improvement in locomotor behaviour, which was significantly (p < 0.05) better than vehicle-treated control animals from the 5th day of treatment onward. c, d Luxol fast blue staining of cross sections of spinal cords from guanosine- (d) and vehicle- (c) treated animals at the lesion site 48 days after the initial lesion (after 14 days treatment) shows increased Luxol fast blue-stained material in d compared to the vehicle-treated control group in c. DC dorsal column at the lesion. Scale bar = 50 μm. e The total area of each section and of the Luxol fast blue-stained portion were measured following the protocol in “Materials and methods”. The quantitative results show that the cords from guanosine-treated animals had significantly greater myelinated area (*p < 0.05), as estimated from the Luxol fast blue staining, than cords from vehicle-treated control animals, which in turn had significantly less myelinated area (**p < 0.001) than unoperated animals. f Correlation between behavioural and histological outcomes was analysed using regression analysis. The data show that the Luxol fast blue-stained area in the injured cords from animals treated with guanosine correlated with their behavioural recovery in open field walking scores, correlation coefficient of 0.72, indicating that the recovery of function may be related to the increase in myelin. Myelin immunofluorescent staining of cross sections of spinal cords from vehicle- (g) and guanosine- (h) treated animals at the lesion site 14 days after treatment shows that guanosine-treated cords (h) contained more myelin basic protein (MBP)-positive profiles from central nervous system at the injured site compared to the vehicle-treated control group (g). IS injured site. Whereas, in i (vehicle-treated cord) and j (guanosine-treated cord) immunolabelling with specific markers for Schwann cell myelin P0 indicated that Schwann cells from the peripheral nervous system did not contribute to guanosine-induced remyelination. Scale bar = 50 μm for all
To determine whether existing cells elaborated the myelin or whether it was a product of other cells that proliferated and subsequently elaborated myelin, the number of proliferating cells was determined by counting all BrdU+ nuclei in cross sections of the lesion penumbra at different times after treatment. In the first 3 days after treatment, cell proliferation at the injury site in both guanosine-treated and control animals was not different from baseline (immediately before treatment; Fig. 2c), although at 3 days there was a trend toward more BrdU+ cells in guanosine-treated animals than controls (112 ± 25 vs 79 ± 16). At 7 and 14 days after guanosine treatment, the number of BrdU+ cells increased significantly; there were 382 ± 42 BrdU+ cells per section at 7 days and 477 ± 49 at 14 days compared with only 88 ± 20 at 7 days and 95 ± 22 at 14 days in controls (p < 0.05; Fig. 2a–c). There were few dividing cells beyond the lesion margins.
Fig. 2Quantitative assessment of immunostaining shows that systemic treatment with guanosine stimulates endogenous oligodendroglial progenitors (NG2+ cells) to proliferate and mature in rats with stable chronic spinal cord injury. a, b Bromodeoxyuridine (BrdU) was used to label proliferating cells after treatment. BrdU immunostaining in cross sections from vehicle- (a) and guanosine- (b) treated animals at the lesion 14 days after treatment shows increased BrdU+ nuclei in b compared to the vehicle-treated control group in a. Scale bar = 50 μm. c Quantitative analysis shows that 7 or 14 days after guanosine treatment there was a significant increase in the number of BrdU+ cells compared with vehicle-treated controls. d, e Demonstrate examples of double-fluorescent immunostaining using antibodies against BrdU (in green) and a marker (NG2) for oligodendroglial progenitors (in red) in cross sections from vehicle- (d) and guanosine- (e) treated animals at the lesion 14 days after treatment. Scale bar = 50 μm. f–h Show quantification of proliferating progenitors (NG2+/BrdU+ double-labelled cells; f) and total number of NG2 cells (g). Data indicate that after 7 or 14 days administration guanosine significantly stimulates proliferation of oligodendroglial progenitors (f, g). Concurrently, guanosine also significantly increases the number of mature oligodendroglia shown in i and j compared to vehicle-treated controls (h, j) using fluorescent immunostaining with a specific marker (Rip) to label the mature oligodendroglia. Scale bar = 50 μm
Oligodendrocytes that survive the demyelinating insult are not thought to be able to contribute to remyelination [41, 42]. Instead, endogenous oligodendrocyte progenitor cells local to the lesion are believed to be the source of new myelinating cells [43]. Previous studies have illustrated that NG2+ cells are the major cycling cell within the resting adult CNS [14, 44] and express many characteristics of oligodendroglial lineage cells, not least their ability to differentiate into oligodendrocytes when isolated into culture. Their role in demyelination and remyelination has been extensively studied and there is evidence that they are the cells responsible for myelin repair [43, 45–48]. To determine whether the proliferating cells were related to NG2+ oligodendroglial progenitor cells, we counted the total number of NG2+ progenitor cells and the number that had proliferated (NG2+/BrdU+) from 2 mm rostral to 2 mm caudal to the lesion site (Fig. 2d,e). There was minimal turnover in the NG2+ cells in the spinal cord during the first 3 days in both guanosine-treated and control animals (13 ± 5 in the guanosine group vs 9 ± 4 cells per section in controls; Fig. 2f). By 7 days, guanosine significantly stimulated progenitor proliferation (85 ± 18 vs 13 ± 4 NG2+/BrdU+ cells/section; Fig. 2f); these cells were observed throughout the penumbra with the majority localised in the dorsal columns. Enhanced proliferation of the NG2+ progenitors after guanosine persisted, and by 14 days, double-labelled progenitors in the guanosine-treated group were eightfold more than in controls (108 ± 12 vs 13 ± 5 NG2+/BrdU+ cells/section; Fig. 2f).
The total number of NG2+ cells was also determined. During the first 3 days, there was no significant difference in the numbers of NG2+ cells in cords at baseline after vehicle treatment or guanosine treatment (Fig. 2g). By 7 days, guanosine treatment increased the total NG2+ cells from 96 ± 22 cells/section at baseline to 266 ± 38 cells/section—more than 2 times higher than in the control group (110 ± 24 /section; Fig. 2g). By 14 days, the numbers of double-labelled progenitors in the cords of guanosine-treated animals were threefold higher than controls (347 ± 36 NG2+ cells/section for guanosine-treated animals; 115 ± 24 cells/section for controls; Fig. 2g).
Mature oligodendrocytes were identified with Rip+ staining (Fig. 2h,i). Guanosine had no effect on mature oligodendroglia 3 days after treatment; at 7 and 14 days, there was a significant increase in Rip+ cells in the penumbra of the cords of animals that had received guanosine compared to controls (7 days: 468 ± 40 vs 205 ± 25; and at 14 days: 492 ± 43 vs 219 ± 23 per section, p < 0.01; Fig. 2j).
Guanosine treatment stimulated astrocyte proliferation in the penumbra as early as 3 days and throughout the 14 days of treatment (Fig. 3a,b) and was detected by antibodies to glial fibrillary acidic protein (GFAP). Astrocytes may stimulate oligodendroglial progenitor proliferation or induce their migration [49]. Guanosine did not alter the numbers of OX-42+ microglia/macrophages until 14 days after treatment. At day 14, guanosine significantly increased OX-42+ cells compared to vehicle treatment (Fig. 3c–e; p < 0.05).
Fig. 3Cross sections of the cords at the injury site staining with GFAP and OX-42 from guanosine-treated and vehicle-treated rats 14 days after treatment following chronic spinal cord injury. a, b Demonstrate examples of GFAP-positive astrocytes (in red) in cross sections from vehicle- (a) and guanosine- (b) treated animals at the injury site 14 days after treatment. c, d Show OX-42-immunofluorescent staining of cross sections of spinal cords from vehicle- (c) and guanosine- (d) treated animals at the lesion site 14 days after treatment. Quantification showed more OX-42-immunolabelled cells in cords of guanosine-treated rats (d, e) compared to cords of vehicle-treated rats (c, e; p < 0.05). Scale bar = 50 μm for all
Guanosine inhibits apoptosis due to a number of stimuli [50–52]. After stroke and spinal cord injury, intrinsic stem cells and progenitors proliferate and differentiate but many die through apoptosis [15, 53–55]. In these experiments, guanosine did not prevent cell death (measured by TUNEL) (Fig. 4), indicating that the protective effects of guanosine do not result from a reduction in precursor cell apoptosis.
Fig. 4Guanosine treatment did not affect apoptosis in the cords with chronic, stable spinal cord injury. For detection of apoptotic cells, a terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labelling (TUNEL) stain was performed using the ‘In situ Cell Death Detection Kit, Fluorescein’. No obvious difference in number of TUNEL-positive cells was observed between vehicle- (a) or guanosine- (b) treated spinal cord sections. Scale bar = 50 μm for all
Before the intraperitoneal injection of guanosine, there were detectable amounts of guanosine and its metabolic product guanine in the spinal cords of injured rats (time 0, Fig. 5a,b). There were no significant differences in guanosine or guanine in the samples of the spinal cords above, at or below the lesion. After guanosine injection, the amount of both guanosine and guanine increased, reaching a maximum by 30 min; the amounts of guanosine and guanine were about twofold and fivefold higher than those detected before the injection (Fig. 5a,b). As with the basal levels (time 0), there was no difference in the amounts of guanosine above, at or below the lesion. Interestingly, guanine levels were always higher than those of guanosine at each of the times tested after injection (Fig. 5a,b).
Fig. 5Guanosine (a) and guanine (b) in spinal cord samples of rats with chronic spinal cord injury before and after intraperitoneal injection of guanosine. Guanosine (8 mg/kg) was administered intraperitoneally to rats 5 weeks after the spinal cord injury. Before or at the indicated time points after the injection, samples of spinal cord from 3–6 animals were taken from the site of the lesion and immediately above and below the lesion. The tissue content of guanosine and its metabolic product guanine was measured by HPLC and CE analysis. Data are the mean ± SEM
New oligodendrocytes in injured spinal cords come from a population of endogenous progenitor cells present in the CNS that can differentiate into mature cells capable of myelinating axons [3, 12, 13, 56]. The data from the present study showed that the increase in the number of NG2+ cells is associated with an increase in Rip+ cells. Our data indicated that guanosine treatment induced an increase of mature oligodendroglia (Rip+ cells), likely due to proliferation and differentiation of their progenitor (NG2+) cells. Therefore, one would expect that Rip+ cells increase after NG2+ cells. However, this was not observed. But guanosine may well stimulate the proliferation of oligodendroglial progenitor cells at several stages of development. This would account for the observed results. Additionally, we cannot exclude that other possible mechanisms are involved in the process; for example, mature oligodendroglia could migrate from the adjacent normal tissue to the injury site soon after guanosine treatment.
Whether the effects on remyelination are due to guanosine, guanine or both is not clear. However, since a putative receptor for guanosine has been identified in the CNS, and since PNP (purine nucleoside phosphorylase) in CNS permits the interconversion of guanine and guanosine enabling guanine to act as a reservoir for guanosine, it is possible that guanosine may be responsible for causing the proliferation of the endogenous progenitor cells and the subsequent remyelination process, although this is by no means certain. But, since guanine itself is difficult to administer because of its poor solubility, administration of guanosine is the most effective delivery mechanism.
The mechanism by which remyelination is facilitated after chronic, traumatic SCI is unknown. Guanosine has been shown to stimulate the production and/or release of a variety of trophic factors [25]; for example bFGF, NGF and NT-3 have been demonstrated to increase the proliferation and survival of oligodendrocyte progenitors [3, 16, 57], induce oligodendroglial genesis [58–61], stimulate the proliferation of oligodendrocyte precursors and enhance remyelination after spinal cord traumatic injury [18, 19] and after toxin-induced demyelination [62]. There are no published reports of the effects of guanine in the vivo systems. However, in cultured cells, guanosine stimulates cell division apparently through a pertussis toxin-sensitive process involving MAP kinase [63]. Moreover, there is evidence of a G protein-coupled receptor in brain membranes that might be a candidate for a guanosine receptor [64]. Further studies will permit us to distinguish amongst these possibilities.
The present findings are the first to show that systemic treatment with guanosine after chronic SCI induces an improvement in function that is accompanied by the formation of mature oligodendrocytes. This effect of guanosine/guanine may be attributable to direct or indirect stimulation of endogenous oligodendrocyte lineage progenitors in the spinal cord and remyelination of axons at the injury site.
These findings raise the possibility that the administration of guanosine may be effective in treating spinal cord injuries and in the treatment of demyelinating diseases such as multiple sclerosis where quiescent progenitors exist in demyelinated plaques [65–70].

myelination
spinal cord
guanosine
oligodendrocyte
cell proliferation
guanine
precursors
functional recovery
immunohistochemistry