Transcriptional analysis of micro-dissected articular cartilage in post-traumatic murine osteoarthritis.

OBJECTIVE: Identify gene changes in articular cartilage of the medial tibial plateau (MTP) at 2, 4 and 8 weeks after destabilisation of the medial meniscus (DMM) in mice. Compare our data with previously published datasets to ascertain dysregulated pathways and genes in osteoarthritis (OA). DESIGN: RNA was extracted from the ipsilateral and contralateral MTP cartilage, amplified, labelled and hybridized on Illumina WGv2 microarrays. Results were confirmed by real-time polymerase chain reaction (PCR) for selected genes. RESULTS: Transcriptional analysis and network reconstruction revealed changes in extracellular matrix and cytoskeletal genes induced by DMM. TGFß signalling pathway and complement and coagulation cascade genes were regulated at 2 weeks. Fibronectin (Fn1) is a hub in a reconstructed network at 2 weeks. Regulated genes decrease over time. By 8 weeks fibromodulin (Fmod) and tenascin N (Tnn) are the only dysregulated genes present in the DMM operated knees. Comparison with human and rodent published gene sets identified genes overlapping between our array and eight other studies. CONCLUSIONS: Cartilage contributes a minute percentage to the RNA extracted from the whole joint (<0.2%), yet is sensitive to changes in gene expression post-DMM. The post-DMM transcriptional reprogramming wanes over time dissipating by 8 weeks. Common pathways between published gene sets include focal adhesion, regulation of actin cytoskeleton and TGFß. Common genes include Jagged 1 (Jag1), Tetraspanin 2 (Tspan2), neuroblastoma, suppression of tumourigenicity 1 (Nbl1) and N-myc downstream regulated gene 2 (Ndrg2). The concomitant genes and pathways we identify may warrant further investigation as biomarkers or modulators of OA.

Effect of γ-cyclodextrin as a lyoprotectant for freeze-dried actinidin.

Actinidin (ATD) is a cysteine protease found in kiwifruit. It is used to tenderize meat and to enhance the digestion of proteins in the small intestine. However, ATD is unstable during freeze-drying, which alters its bioactivity. It is well known that sugars have the ability to protect proteins from the stress of freeze-drying. In this study, we investigated the protective effect of various saccharides on the stability of ATD during freeze-drying. The ATD activities of the samples containing γ-cyclodextrin (CyD) showed only a small decrease, and compared with trehalose and sucrose, γ-CyD was a more effective stabilizer for ATD. Secondary structural changes in freeze-dried ATD were observed by circular dichroism spectroscopy and compared with the changes in stabilized samples. There was a close relationship between the α-helix content and the stabilization. The sugars stabilized the protein by suppressing the changes in the α-helix. Fourier transform infrared spectroscopy measurement showed that the amide I band of ATD with γ-CyD was shifted to a lower wavenumber compared with other sugars. Therefore, stronger hydrogen bonds may be formed between ATD and γ-CyD than between ATD and other sugars. The suppression of changes in the protein secondary structure accompanying the formation of hydrogen bonding between the protein and the sugar also contributed to the protective effect of the sugars.

In vivo imaging of MMP-13 activity in the murine destabilised medial meniscus surgical model of osteoarthritis.

OBJECTIVE: To detect and determine disease severity of osteoarthritis (OA) using a probe activated by matrix metalloproteinase-13 (MMP-13) in vivo in the murine destabilised medial meniscus (DMM) surgical model of OA. DESIGN: We have previously described MMP12ap and MMP13ap, internally quenched fluorescent peptide substrate probes that are activated respectively by MMP-12 and MMP-13. Here we used these probes to follow enzyme activity in vivo in mice knees 4, 6 and 8 weeks following DMM surgery. After in vivo optical imaging, disease severity was determined through traditional histological analysis. The amount of probe activation was analysed for discrimination between DMM, contralateral and sham operated knees, as well as for congruence between activity and histological damage. RESULTS: There was no specific activation of MMP12ap at the time points observed between sham operated and DMM operated, or their respective contralateral joints. The activation of the MMP13ap in the DMM model was highest 6 weeks after surgery, but was only specific compared against sham surgery 8 weeks after surgery (1.5-fold increase). The activation of MMP13ap correlated with histological damage 6 and 8 weeks after surgery, with correlations of 0.484 (P = 0.0032) and 0.478 respectively (P = 0.0049). This correlation dropped to 0.218 (P = 0.011) if all data were considered. CONCLUSION: The current MMP-13 activity probe is suitable for the discrimination between DMM and sham or contralateral knees 8 weeks after surgery, when cartilage loss is typified by the appearance of small fissures up to the tidemark, but not earlier. This activity correlates with the histological damage observed.

Deiodinase 2 upregulation demonstrated in osteoarthritis patients cartilage causes cartilage destruction in tissue-specific transgenic rats.

OBJECTIVE: Chondrocyte hypertrophy followed by cartilage destruction is a crucial step for osteoarthritis (OA) development, however, the underlying mechanism remains largely unknown. The objectives of this study are to identify the gene that may cause cartilage hypertrophy and to elucidate its role on OA pathogenesis. DESIGN: Gene expression profiles of cartilages from OA patients and normal subjects were examined by microarray analysis. Expression of deiodinases, enzymes for regulation of triiodothyronine (T3) biosynthesis, in human and rat articular cartilage (AC) were examined by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Rat ACs and chondrocytes were treated with T3 to investigate its role on chondrocyte hypertrophy and inflammatory reaction. Cartilage-specific Type II deiodinase (DIO2) transgenic rats were generated using bacterial artificial chromosome harboring the entire rat Col2a1 and human DIO2 gene. An experimental OA model was created in the animal to examine the role of DIO2 on cartilage degeneration. RESULTS: DIO2 is highly expressed in OA patient AC compared to normal control. In rat AC, DIO2 is specifically expressed among deiodinases and dominantly expressed the same as in brown adipose tissue. T3 induces hypertrophic markers in articular chondrocyte and cartilage explant culture, and enhances the effect of IL-1α on induction of cartilage degrading enzymes. Importantly, cartilage-specific DIO2 transgenic rats are more susceptible to knee joint destabilization and develop severe AC destruction. CONCLUSION: Our findings demonstrate that upregulated expression of DIO2 in OA patient cartilage might be responsible for OA pathogenesis by enhancing the chondrocyte hypertrophy and inflammatory response.

Distinct genetic loci control development of benign and malignant skin tumours in mice.

Genetic susceptibility to chemically induced skin cancer in mice is controlled by multiple unlinked genetic loci. Mus spretus mice have dominant resistance genes which confer resistance to interspecific F1 hybrids with susceptible Mus musculus strains. We have mapped three major resistance loci using a combination of Mapmaker/QTL analysis and multiple regression analysis to mouse chromosomes 5 and 7. At least two independent loci on chromosome 7 exert their effects primarily during benign tumour development and have very little influence on tumour progression. On the other hand, probably a single locus on chromosome 5 affects both early and late stages of malignancy. The results indicate that benign and malignant tumours are largely under independent genetic control.

Effect of κ-opioid receptor agonist on the growth of non-small cell lung cancer (NSCLC) cells.

BACKGROUND: It is becoming increasingly recognised that opioids are responsible for tumour growth. However, the effects of opioids on tumour growth have been controversial. METHODS: The effects of κ-opioid receptor (KOR) agonist on the growth of non-small cell lung cancer (NSCLC) cells were assessed by a cell proliferation assay. Western blotting was performed to ascertain the mechanism by which treatment with KOR agonist suppresses tumour growth. RESULTS: Addition of the selective KOR agonist U50,488H to gefitinib-sensitive (HCC827) and gefitinib-resistant (H1975) NSCLC cells produced a concentration-dependent decrease in their growth. These effects were abolished by co-treatment with the selective KOR antagonist nor-BNI. Furthermore, the growth-inhibitory effect of gefitinib in HCC827 cells was further enhanced by co-treatment with U50,488H. With regard to the inhibition of tumour growth, the addition of U50, 488H to H1975 cells produced a concentration-dependent decrease in phosphorylated-glycogen synthase kinase 3ß (p-GSK3ß). CONCLUSION: The present results showed that stimulation of KOR reduces the growth of gefitinib-resistant NSCLC cells through the activation of GSK3ß.

Involvement of ADAMTS5 and hyaluronidase in aggrecan degradation and release from OSM-stimulated cartilage.

The relative contribution of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4 and ADAMTS5 to aggrecan degradation under oncostatin M (OSM) stimulation, the role of the ancillary domains of the aggrecanases on their ability to cleave within the chondroitin sulfate (CS)-2 region, the role of hyaluronidases (HYAL) in stimulating aggrecan release in the absence of proteolysis, and the identity of the hyaluronidase involved in OSM-mediated cartilage breakdown were investigated. Bovine articular cartilage explants were cultured in the presence of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha) and/or OSM, or treated with trypsin and/or hyaluronidase. Aggrecan was digested with various domain-truncated isoforms of ADAMTS4 and ADAMTS5. Aggrecan and link protein degradation and release were analyzed by immunoblotting. Aggrecanase and HYAL gene expression were determined. ADAMTS4 was the most inducible aggrecanase upon cytokine stimulation, whereas ADAMTS5 was the most abundant aggrecanase. ADAMTS5 was the most active aggrecanase and was responsible for the generation of an OSM-specific degradation pattern in the CS-2 region. Its ability to cleave at the OSM-specific site adjacent to the aggrecan G3 region was enhanced by truncation of the C-terminal thrombospondin domain, but reduced by further truncation of both the spacer and cysteine-rich domains of the enzyme. OSM has the ability to mediate proteoglycan release through hyaluronan degradation, under conditions where HYAL-2 is the predominant hyaluronidase being expressed. Compared to other catabolic cytokines, OSM exhibits a unique potential at degrading the proteoglycan aggregate, by promoting early robust aggrecanolysis, primarily through the action of ADAMTS5, and hyaluronan degradation.

Effect of tiotropium bromide on airway inflammation and remodelling in a mouse model of asthma.

BACKGROUND: Tiotropium bromide, a long acting muscarinic receptor inhibitor, is a potent agent for patients with bronchial asthma as well as chronic obstructive pulmonary disease. OBJECTIVE: The aim of this study was to evaluate whether tiotropium bromide can inhibit allergen-induced acute and chronic airway inflammation, T helper (Th)2 cytokine production, and airway remodelling in a murine model of asthma. METHODS: Balb/c mice were sensitized and challenged acutely or chronically to ovalbumin (OVA). The impact of tiotropium bromide was assessed using these mice models by histologic, morphometric, and molecular techniques. Moreover, the effect of tiotropium bromide on Th2 cytokine production from purified human peripheral blood mononuclear cells (PBMCs) was assessed. RESULTS: Treatment with tiotropium bromide significantly reduced airway inflammation and the Th2 cytokine production in bronchoalveolar lavage fluid (BALF) in both acute and chronic models of asthma. The levels of TGF-beta1 were also reduced by tiotropium bromide in BALF in a chronic model. The goblet cell metaplasia, thickness of airway smooth muscle, and airway fibrosis were all significantly decreased in tiotropium bromide-treated mice. Moreover, airway hyperresponsiveness (AHR) to serotonin was significantly abrogated by tiotropium bromide in a chronic model. Th2 cytokine production from spleen cells isolated from OVA-sensitized mice was also significantly inhibited by tiotropium bromide and 4-diphenylacetoxy-N-methylpiperidine methiodide, which is a selective antagonist to the M3 receptor. Finally, treatment with tiotropium bromide inhibited the Th2 cytokine production from PBMCs. CONCLUSION: These results indicate that tiotropium bromide can inhibit Th2 cytokine production and airway inflammation, and thus may reduce airway remodelling and AHR in a murine model of asthma.

Interleukin 13 (IL-13)-regulated expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging cartilage.

OBJECTIVE: The adamalysin metalloproteinase 15 (ADAM15) has been shown to protect against development of osteoarthritis in mice. Here, we have investigated factors that control ADAM15 levels in cartilage. DESIGN: Secretomes from wild-type and Adam15 -/- chondrocytes were compared by label-free quantitative mass spectrometry. mRNA was isolated from murine knee joints, either with or without surgical induction of osteoarthritis on male C57BL/6 mice, and the expression of Adam15 and other related genes quantified by RT-qPCR. ADAM15 in human normal and osteoarthritic cartilage was investigated similarly and by fluorescent immunohistochemistry. Cultured HTB94 chondrosarcoma cells were treated with various anabolic and catabolic stimuli, and ADAM15 mRNA and protein levels evaluated. RESULTS: There were no significant differences in the secretomes of chondrocytes from WT and Adam15 -/- cartilage. Expression of ADAM15 was not altered in either human or murine osteoarthritic cartilage relative to disease-free controls. However, expression of ADAM15 was markedly reduced upon aging in both species, to the extent that expression in joints of 18-month-old mice was 45-fold lower than in that 4.5-month-old animals. IL-13 increased expression of ADAM15 in HTB94 â€‹cells by 2.5-fold, while modulators of senescence and autophagy pathways had no effect. Expression of Il13 in the joint was reduced with aging, suggesting this cytokine may control ADAM15 levels in the joint. CONCLUSION: Expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging human and murine joints, possibly due to a concomitant reduction in IL-13 expression. We thus propose IL-13 as a novel factor contributing to increased osteoarthritis risk upon aging.

Covalent modification of matrix metalloproteinases by a photoaffinity probe: influence of nucleophilicity and flexibility of the residue in position 241.

A photoaffinity probe, developed for the specific labeling of matrix metalloproteinase (MMP) active sites, was recently shown to covalently modify a single residue in human MMP-12, namely, Lys(241), by reacting selectively with the side chain epsilon-amino group of that residue. The residue in position 241 of MMPs is not conserved; thus, variability in this position may be responsible for the dispersion in cross-linking yield observed between MMPs when labeled by this photoaffinity probe. By studying the pH dependence of the labeling properties of this probe toward different MMPs (MMP-12, MMP-3, MMP-9, and various mutants of human MMP-12) and identifying the site of covalent modification of MMP-3 by this probe, our new data demonstrated that the nucleophilicity of the residue in position 241 plays a key role in determining the cross-linking yield of MMP modification by the probe. However, these studies also reveal that subtle additional structural parameters, including local conformation and flexibility, of the residue in position 241 should also be taken into consideration, a property adding a further degree of complexity in our understanding of the photolabeling probe reactivity and in designing optimal photoaffinity probes for performing functional proteomic studies of zinc proteinases like MMPs.

Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.

We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

Convergence of congenic mapping and allele-specific alterations in tumors for the resolution of the Skts1 skin tumor susceptibility locus.

Although several familial cancer genes with high-penetrance mutations have been identified, the major genetic component of susceptibility to sporadic cancers is attributable to low-penetrance alleles. These 'weak' tumor susceptibility genes do not segregate as single Mendelian traits and are therefore difficult to find in studies of human populations. Previously, we have proposed that a combination of germline mapping and analysis of allele-specific imbalance in tumors may be used to refine the locations of susceptibility genes using mouse models of cancer. Here, we have used linkage analysis and congenic mouse strains to map the major skin tumor susceptibility locus Skts1 within a genetic interval of 0.9 cM on proximal chromosome 7. This interval lies in an apparent recombination cold spot, and corresponds to a physical distance of about 15 Mb. We therefore, used patterns of allele-specific imbalances in tumors from backcross and congenic mice to refine the location of Skts1. We demonstrate that this single tumor modifier locus has a dramatic effect on the allelic preference for imbalance on chromosome 7, with at least 90% of tumors from the congenics showing preferential gain of markers on the chromosome carrying the susceptibility variant. Importantly, these alterations enabled us to refine the location of Skts1 at higher resolution than that attained using the congenic mice. We conclude that low-penetrance susceptibility genes can have strong effects on patterns of allele-specific somatic genetic changes in tumors, and that analysis of the directionality of these somatic events provides an important and rapid route to identification of germline genetic variants that confer increased cancer risk.

Characterization of an ADAMTS-5-mediated cleavage site in aggrecan in OSM-stimulated bovine cartilage.

OBJECTIVE: In a previous study, we identified a 50-kDa G3-containing aggrecan degradation product in bovine cartilage, released from the tissue after interleukin-1 (IL-1) stimulation in the presence of oncostatin M (OSM). Our objective was to purify, determine the N-terminal sequence of this fragment and verify whether this cleavage could be attributed to a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 action in vitro. METHODS: Collected media from bovine cartilage explant cultures stimulated with IL-1+OSM were subjected to anion-exchange chromatography. The N-terminal sequence of the fragment of interest in the purified fractions was determined by automated Edman sequencing. Fetal bovine aggrecan was digested with full-length recombinant ADAMTS-4 and ADAMTS-5 and resulting degradation products were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and immunoblotting using an anti-G3 antiserum and an anti-neoepitope antibody that had been generated to the new N-terminus of the G3 fragment. RESULTS: Characterization of the 50-kDa fragment showed that it possesses chondroitin sulfate (CS) and is the result of a cleavage within the C-terminal portion of the CS-2 domain, adjacent to the G3 region. Sequence analysis identified the cleavage region as TQRPAE(2047)-(2048)ARLEIE, suggesting an aggrecanase-derived product. Using an anti-neoepitope antibody specific for the additional cleavage site, it was shown that the product is generated in vitro upon digestion of aggrecan by ADAMTS-5 and, to a much lesser extent, by ADAMTS-4. CONCLUSIONS: The abundance and rapid rate of release of this degradation product in organ cultures in the presence of OSM suggest that it could result from a unique aggrecan proteolysis mediated by aggrecanases.

Crystal structure of an anhydrous form of trehalose: structure of water channels of trehalose polymorphism.

alpha, alpha-Trehalose (trehalose) is a nonreducing disaccharide of glucose and is accumulated at high concentrations in some anhydrobiotic organisms, which can survive without water for long periods and rapidly resume active metabolism upon hydration. Although it has been proposed that the intriguing mechanism of bioprotection in anhydrobiosis is conferred by a water channel, details of such a channel have yet to be revealed. We determined the crystal structure of a trehalose anhydrate to further understand the relationship between the structure of water channels and the trehalose polymorph. The space group was identical to that of the dihydrate and the lattice constants were also very similar. Among the five intermolecular hydrogen bonds between the trehalose molecules, four were preserved in the anhydrate. If dehydration of the dihydrate is slow and/or gentle enough to preserve the hydrogen bonds, transformation from the dihydrate to the anhydrate may occur. There are two different holes, hole-1 and hole-2, along one crystal axis. Hole-1 is constructed by trehalose molecules with a screw diad at its center, while hole-2 has a smaller diameter and is without a symmetry operator. Because of the screw axis at the center of hole-1, hollows are present at the side of the hole with diameters roughly equal to that of hole-1. Hole-1 and side pockets followed by hollows correspond to the positions of two water molecules of the dihydrate. The side hollows of the water channel are also observed in the water-filled hole of the dihydrate. Consequently, hole-1 is considered to be a one-dimensional water channel with side pockets. We also calculated molecular and crystal energies to examine the rapid water uptake of the anhydrate. It was demonstrated that the intermolecular interactions in the anhydrate were weaker than in the other anhydrous form, and probably also than those in amorphous trehalose. The anhydrate provides water capture for another solid form and gives protection from water uptake. These structural properties of the anhydrate may elucidate bioprotection in anhydrobiosis.

Improvement of solubility of C70 by complexation with cyclomaltononaose (delta-cyclodextrin).

We investigated the solubilizing effects of cyclomaltononaose (delta-CD), a cyclic oligosaccharide composed of nine alpha-1,4-linked D-glucose units, on C70 by using the ball-milling method based on a solid-solid mechanochemical reaction. The complex between C70 and delta-CD was characterized by UV-VIS spectrometry and fast atom bombardment mass spectrometry (FAB-MS). Coloration of the C70/delta-CD system was red-brown in aqueous solution, and the UV-VIS spectrum was in agreement with that of C70 in hexane solution. The FAB-MS spectrum of the C70/delta-CD system showed a negative ion peak corresponding to the molecular weight of a complex between two delta-CD and one C70. These findings suggest that the solubilization of C70 in water was due to complex formation of C70 with delta-CD, and the stoichiometric ratio of this complex was 1: 2 (C70: delta-CD).

Cancer resistance genes in mice: models for the study of tumour modifiers.

Smokers have frequently been heard to defend their habit by recounting anecdotes about relatives or friends who have smoked heavily for many years without developing cancer. While individuals who have survived many years of repeated mutagen exposure are probably very rare, their existence suggests that some people are intrinsically resistant to the effects of carcinogens, probably because of their genetic background. This interpretation is supported by studies on mouse strains that are highly resistant to the development of tumours induced by treatment with exogenous carcinogens. In this review we discuss the advantages of the mouse as a model system for the isolation of cancer-resistance genes that have potentially important uses in diagnostics, prevention and tumour therapy.

Synovial procollagenase activation by human mast cell tryptase dependence upon matrix metalloproteinase 3 activation.

Mast cells have been implicated in the pathogenesis of the matrix degradation observed in the cartilaginous and osseous structures of the rheumatoid joint. We previously reported that human mast cell tryptase, a 134-kD granule-associated neutral protease, is present in rheumatoid synovium and can activate collagenase in crude culture medium in vitro. the present study attempts to depict the precise mechanism of this activation. To express full activation of latent collagenase, matrix metalloproteinase 3 (MMP-3) or stromelysin, can be activated by tryptase in a time and dose-dependent manner. Tryptase was not capable of generating active collagenase in the crude media from cultured rheumatoid synoviocytes depleted of proMMP-3 by immunoadsorption. In addition, the function of the tissue inhibitor of metalloproteinases (TIMP) was not altered by tryptase, and SDS-PAGE analysis revealed no degradation of TIMP by tryptase. The tryptase dependent activation of synoviocyte procollagenase thereby appears to be entirely dependent upon its ability to activate proMMP-3.

Neuroprotective effect of erythropoietin, and role of metallothionein-1 and -2, in permanent focal cerebral ischemia.

Metallothioneins (MTs) are small cysteine-rich proteins found widely throughout the mammalian body, including the CNS. MT-1 and -2 protect against reactive oxygen species and free radicals. We investigated the role of MT-1 and -2 using MT-1,-2 knockout (KO) mice. MT-1,-2 KO mice exhibited greater neuronal damage after permanent middle cerebral artery occlusion (MCAO) than wild-type mice. MT-2 mRNA was significantly increased at 6, 12, and 24 h after MCAO in the wild-type mouse brain [as detected by real-time reverse-transcription polymerase chain reaction (RT-PCR)], while MT-1 and MT-3 were decreased at 12 and 24 h. In an immunohistochemical study, MT expression displayed colocalization with glial fibrillary acidic protein (GFAP)-positive cells (astrocytes) in the penumbra area in wild-type mice. Since erythropoietin (EPO) has been reported to induce MT-1 and -2 gene expression in vitro, we examined its effect after permanent MCAO, and explored the possible underlying mechanism by examining MT-1 and -2 induction in vivo. In wild-type mice, EPO significantly reduced both infarct area and volume at 24 h after the ischemic insult. However, in MT-1,-2 KO mice EPO-treatment did not alter infarct volume (vs. vehicle-treatment). In wild-type mice at 6 h after EPO administration, real-time RT-PCR revealed increased MT-1 and -2 mRNA expression in the cerebral cortex (without MCAO). Further, MT-1 and -2 immunoreactivity was increased in the cortex of EPO-treated mice. These findings indicate that MTs are induced, and may be neuroprotective against neuronal damage, after MCAO. Furthermore, EPO is neuroprotective in vivo during permanent MCAO, and this may be at least partly mediated by MTs.

Gene expression levels of gamma-glutamyl hydrolase in tumor tissues may be a useful biomarker for the proper use of S-1 and tegafur-uracil/leucovorin in preoperative chemoradiotherapy for patients with rectal cancer.

PURPOSE: Preoperative chemoradiotherapy (CRT) using 5-fluorouracil (5-FU)-based chemotherapy is the standard of care for rectal cancer. The effect of additional chemotherapy during the period between the completion of radiotherapy and surgery remains unclear. Predictive factors for CRT may differ between combination chemotherapy with S-1 and with tegafur-uracil/leucovorin (UFT/LV). METHODS: The subjects were 54 patients with locally advanced rectal cancer who received preoperative CRT with S-1 or UFT/LV. The pathological tumor response was assessed according to the tumor regression grade (TRG). The expression levels of 18 CRT-related genes were determined using RT-PCR assay. RESULTS: A pathological response (TRG 1-2) was observed in 23 patients (42.6%). In a multivariate logistic regression analysis for pathological response, the overall expression levels of four genes, HIF1A, MTHFD1, GGH and TYMS, were significant, and the accuracy rate of the predictive model was 83.3%. The effects of the gene expression levels of GGH on the response differed significantly according to the treatment regimen. The total pathological response rate of both high-GGH patients in the S-1 group and low-GGH patients in the UFT/LV group was 58.3%. CONCLUSION: Additional treatment with 5-FU-based chemotherapy during the interval between radiotherapy and surgery is not beneficial in patients who have received 5-FU-based CRT. The expression levels of four genes, HIF1A, MTHFD1, GGH and TYMS, in tumor tissues can predict the response to preoperative CRT including either S-1 or UFT/LV. In particular, the gene expression level of GGH in tumor tissues may be a useful biomarker for the appropriate use of S-1 and UFT/LV in CRT.