Heparan sulfate deficiency leads to hypertrophic chondrocytes by increasing bone morphogenetic protein signaling.

OBJECTIVE: Exostosin-1 (EXT1) and EXT2 are the major genetic etiologies of multiple hereditary exostoses and are essential for heparan sulfate (HS) biosynthesis. Previous studies investigating HS in several mouse models of multiple hereditary exostoses have reported that aberrant bone morphogenetic protein (BMP) signaling promotes osteochondroma formation in Ext1-deficient mice. This study examined the mechanism underlying the effects of HS deficiency on BMP/Smad signaling in articular cartilage in a cartilage-specific Ext-/- mouse model. METHOD: We generated mice with a conditional Ext1 knockout in cartilage tissue (Ext1-cKO mice) using Prg4-Cre transgenic mice. Structural cartilage alterations were histologically evaluated and phospho-Smad1/5/9 (pSmad1/5/9) expression in mouse chondrocytes was analyzed. The effect of pharmacological intervention of BMP signaling using a specific inhibitor was assessed in the articular cartilage of Ext1-cKO mice. RESULTS: Hypertrophic chondrocytes were significantly more abundant (P = 0.021) and cartilage thickness was greater in Ext1-cKO mice at 3 months postnatal than in control littermates (P = 0.036 for femur; and P < 0.001 for tibia). However, osteoarthritis did not spontaneously occur before the 1-year follow-up. matrix metalloproteinase (MMP)-13 and adamalysin-like metalloproteinases with thrombospondin motifs(ADAMTS)-5 were upregulated in hypertrophic chondrocytes of transgenic mice. Immunostaining and western blotting revealed that pSmad1/5/9-positive chondrocytes were more abundant in the articular cartilage of Ext1-cKO mice than in control littermates. Furthermore, the BMP inhibitor significantly decreased the number of hypertrophic chondrocytes in Ext1-cKO mice (P = 0.007). CONCLUSIONS: HS deficiency in articular chondrocytes causes chondrocyte hypertrophy, wherein upregulated BMP/Smad signaling partially contributes to this phenotype. HS might play an important role in maintaining the cartilaginous matrix by regulating BMP signaling.

The Geriatric Nutritional Risk Index is Independently Associated with Prognosis in Patients with Critical Limb Ischemia Following Endovascular Therapy.

OBJECTIVES: Patients with critical limb ischemia (CLI) have poor overall and limb prognosis. Although nutritional status influences overall prognosis, and the Geriatric Nutritional Risk Index (GNRI) is a widely used, simple and well established nutritional status screening method, the association between the GNRI and the overall and limb prognosis of patients with CLI following endovascular therapy (EVT) has not been explored. METHODS: Clinical outcomes were retrospectively evaluated in 473 consecutive patients (74 ± 10 years; 59% male) with CLI who underwent EVT. The GNRI on admission was calculated as follows: [14.89 × albumin (g/dL)] + [41.7 × (body weight/ideal body weight)]. Cox proportional hazard analysis was performed to explore the independent association between the GNRI and mortality and major amputation. RESULTS: Patients (53% ambulatory, 38% wheelchair bound, and 9% bedridden) were divided into two groups based on the median GNRI: the higher group (GNRI ≥ 91.2, n = 237) and the lower group (GNRI < 91.2, n = 236). Median follow up duration after EVT was 11.3 months. Three years after EVT, the survival rate (74% in the higher GNRI, and 48% in the lower GNRI, respectively), and limb salvage rate (92% in the higher GNRI, and 84% in the lower GNRI) were significantly lower in the lower GNRI group. GNRI (hazard ratio [HR], 1.03; 95% confidence interval [CI], 1.01-1.05), along with being wheelchair bound (HR, 1.87; 95% CI 1.17-2.97; vs. ambulatory status), being bedridden (HR, 3.10; 95% CI, 1.63-2.97; vs. ambulatory status), being on hemodialysis (HR, 2.33; 95% CI, 1.49-3.64), and having chronic heart failure (HR, 2.22; 95% CI, 1.44-3.43) were the independent predictors of mortality. The GNRI (HR, 1.04; 95% CI, 1.01-1.07), being bedridden (HR, 4.15; 95% CI, 1.67-10.3; vs. ambulatory status), isolated below knee disease (HR, 2.49; 95% CI, 1.30-4.77), and hemodialysis (HR, 2.44; 95% CI, 1.23-4.85) were independently associated with major amputation. CONCLUSIONS: The GNRI on admission was independently associated with mortality and major amputation after EVT in patients with CLI.

High-Convexity Tightness Predicts the Shunt Response in Idiopathic Normal Pressure Hydrocephalus.

BACKGROUND AND PURPOSE: Although neuroimaging plays an important role in the diagnosis of idiopathic normal pressure hydrocephalus, its predictive value for response to shunt surgery has not been established. The purpose of the current study was to identify neuroimaging markers that predict the shunt response of idiopathic normal pressure hydrocephalus. MATERIALS AND METHODS: Sixty patients with idiopathic normal pressure hydrocephalus underwent presurgical brain MR imaging and clinical evaluation before and 1 year after shunt surgery. The assessed MR imaging features included the Evans index, high-convexity tightness, Sylvian fissure dilation, callosal angle, focal enlargement of the cortical sulci, bumps in the lateral ventricular roof, and deep white matter and periventricular hyperintensities. The idiopathic normal pressure hydrocephalus grading scale total score was used as a primary clinical outcome measure. We used measures for individual symptoms (ie, the idiopathic normal pressure hydrocephalus grading scale subdomain scores, such as gait, cognitive, and urinary scores), the Timed Up and Go test, and the Mini-Mental State Examination as secondary clinical outcome measures. The relationships between presurgical neuroimaging features and postoperative clinical changes were investigated by using simple linear regression analysis. To identify the set of presurgical MR imaging features that best predict surgical outcomes, we performed multiple linear regression analysis by using a bidirectional stepwise method. RESULTS: Simple linear regression analyses demonstrated that presurgical high-convexity tightness, callosal angle, and Sylvian fissure dilation were significantly associated with the 1-year changes in the clinical symptoms. A multiple linear regression analysis demonstrated that presurgical high-convexity tightness alone predicted the improvement of the clinical symptoms 1 year after surgery. CONCLUSIONS: High-convexity tightness is a neuroimaging feature predictive of shunt response in idiopathic normal pressure hydrocephalus.

Tumour-suppressive microRNA-144-5p directly targets CCNE1/2 as potential prognostic markers in bladder cancer.

BACKGROUND: Analysis of a microRNA (miRNA) expression signature of bladder cancer (BC) by deep-sequencing revealed that clustered miRNAs microRNA (miR)-451a, miR-144-3p, and miR-144-5p were significantly downregulated in BC tissues. We hypothesised that these miRNAs function as tumour suppressors in BC. The aim of this study was to investigate the functional roles of these miRNAs and their modulation of cancer networks in BC cells. METHODS: The functional studies of BC cells were performed using transfection of mature miRNAs. Genome-wide gene expression analysis, in silico analysis, and dual-luciferase reporter assays were applied to identify miRNA targets. The association between miR-144-5p levels and expression of the target genes was determined, and overall patient survival as a function of target gene expression was estimated by the Kaplan-Meier method. RESULTS: Gain-of-function studies showed that miR-144-5p significantly inhibited cell proliferation by BC cells. Four cell cycle-related genes (CCNE1, CCNE2, CDC25A, and PKMYT1) were identified as direct targets of miR-144-5p. The patients with high CCNE1 or CCNE2 expression had lower overall survival probabilities than those with low expression (P=0.025 and P=0.032). CONCLUSION: miR-144-5p functions as tumour suppressor in BC cells. CCNE1 and CCNE2 were directly regulated by miR-144-5p and might be good prognostic markers for survival of BC patients.

Characterization of ASKP1240, a fully human antibody targeting human CD40 with potent immunosuppressive effects.

Blocking the CD40-CD154 interaction is reported to be effective for transplantation management and autoimmune disease models in rodents and nonhuman primates. However, clinical trials with anti-CD154 mAbs were halted because of high incidence of thromboembolic complications. Thus, we generated and characterized a fully human anti-CD40 mAb ASKP1240, as an alternative to anti-CD154 mAb. In vitro ASKP1240 concentration-dependently inhibited human peripheral blood mononuclear cell proliferation induced by soluble CD154. In addition, ASKP1240 did not destabilize platelet thrombi under physiological high shear conditions while mouse anti-human CD154 mAb (mu5C8) did. And ASKP1240 itself did not activate platelet and endothelial cells. In vivo administration of ASKP1240 (1 or 10 mg/kg, intravenously) to cynomolgus monkeys, weekly for 3 weeks, significantly attenuated both delayed-type hypersensitivity and specific antibody formation evoked by tetanus toxoid. The immunosuppressive effect was well correlated with the CD40 receptor saturation. Thus, these results suggest that ASKP1240 is immunosuppressive but not prothromboembolic, and as such appears to be a promising therapeutic candidate for the management of solid organ transplant rejection and autoimmune diseases therapy.

Role of c-Jun N-terminal kinase isoforms in the cellular activity of melanoma cell lines.

BACKGROUND: The c-Jun N-terminal kinase (JNK) is thought to be involved in inflammation, proliferation and apoptosis. AIM: To examine the role of JNK isoforms in metastasis, proliferation, migration and invasion of the malignant melanoma (MM) cell lines SK-MEL-28, SK-MEL-3 and WM164, using a kinase-specific inhibitor or isoform-specific small interfering (si)RNAs. RESULTS: SK-MEL-3, a cell line established from metastatic MM, showed slightly increased phosphorylation of both JNK1 and JNK2, whereas WM164, a cell line derived from primary MM, showed significant phosphorylation of JNK1. A JNK inhibitor, SP600125, inhibited cell proliferation of SK-MEL-3 but not SK-MEL-28 or WM164. Transfection of JNK1-specific siRNA reduced the migratory activity of WM164 cells, while silencing of either JNK1 or JNK2 strongly suppressed the invasive activity of SK-MEL-3. CONCLUSIONS: Our study suggests that JNK isoforms have different roles in MM. Metastasis of MM may be regulated by JNK2, while invasion is regulated by both JNK1 and JNK2. JNK1 and JNK2 respectively mediate cell migration and cell proliferation. Further understanding of the specific roles of JNK isoforms in the pathogenesis of MM may lead to the development of therapies targeting specific isoforms.

Stabilization of phenotypic plasticity through mesenchymal-specific DNA hypermethylation in cancer cells.

The epithelial-mesenchymal transition (EMT) has a crucial role in normal and disease processes including tumor progression. In this study, we first classified epithelial-like and mesenchymal-like oral squamous cell carcinoma (OSCC) cell lines based on expression profiles of typical EMT-related genes using a panel of 18 OSCC cell lines. Then, we performed methylation-based and expression-based analyses of components of the Wnt signaling pathway, and identified WNT7A and WNT10A as genes silenced by mesenchymal-specific DNA hypermethylation in OSCCs. A significant association was revealed between some clinicopathological findings and the DNA methylation status of WNT7A (normal vs tumor, P=0.007; T1-2 vs T3-4, P=0.040; I-III vs IV, P=0.016) and WNT10A (N0-N1 vs N2-N3, P=0.046) in the advanced stages of OSCC. Moreover, we found that E-cadherin expression in cancer cells may be positively regulated by WNT7A, whose expression is negatively regulated by mesenchymal-specific DNA hypermethylation or ZEB1 in mesenchymal-like OSCC cells. Our findings indicate that epithelial-specific gene silencing through mesenchymal-specific DNA hypermethylation may stabilize the phenotypic plasticity of cancer cells during EMT/MET.

Increased circulating cell signalling phosphoproteins in sera are useful for the detection of pancreatic cancer.

BACKGROUND: Intracellular phosphoprotein activation significantly regulates cancer progression. However, the significance of circulating phosphoproteins in the blood remains unknown. We investigated the serum phosphoprotein profile involved in pancreatic cancer (PaCa) by a novel approach that comprehensively measured serum phosphoproteins levels, and clinically applied this method to the detection of PaCa. METHODS: We analysed the serum phosphoproteins that comprised cancer cellular signal pathways by comparing sera from PaCa patients and benign controls including healthy volunteers (HVs) and pancreatitis patients. RESULTS: Hierarchical clustering analysis between PaCa patients and HVs revealed differential pathway-specific profiles. In particular, the components of the extracellular signal-regulated kinase (ERK) signalling pathway were significantly increased in the sera of PaCa patients compared with HVs. The positive rate of p-ERK1/2 (82%) was found to be superior to that of CA19-9 (53%) for early stage PaCa. For the combination of these serum levels, the area under the receiver-operator characteristics curves was showing significant ability to distinguish between the two populations in independent validation set, and between cancer and non-cancer populations in another validation set. CONCLUSION: The comprehensive measurement of serum cell signal phosphoproteins is useful for the detection of PaCa. Further investigations will lead to the implementation of tailor-made molecular-targeted therapeutics.

Extensive multiple piloleiomyoma in the submental region treated successfully by surgery and reconstruction with a submental perforator flap.

Piloleiomyoma is a benign tumour originating in the smooth muscles of the arrector pili muscle in the skin. The lesions are often sensitive to touch, cold and emotional disturbance. We present a patient with multiple piloleiomyoma (MPL) of the submentum who underwent reconstructive surgery using a submental perforator flap. The result was excellent and there were no postoperative complications. MPL of the submental region is relatively rare; to our knowledge, ours is the first report of MPL treated successfully with a submental perforator flap.

Induction of murine AA amyloidosis by various homogeneous amyloid fibrils and amyloid-like synthetic peptides.

We investigated amyloid-enhancing factor (AEF) activity of amyloid fibrils extracted from amyloid-laden livers of mice, cow, cheetah, cat and swan. All amyloid fibrils were confirmed to be amyloid protein A (AA) by an immunohistochemical analysis. We found that these fibrils accelerated the deposition of amyloid in an experimental mouse model of AA amyloidosis. Furthermore, the degree of deposition was dependent on the concentration of fibrils. When we compared the minimal concentration of amyloid fibrils needed to induce deposition, we found that these fibrils showed different efficiencies. Murine amyloid fibril induced amyloid deposition more efficiently than cow, cat, cheetah or swan amyloid fibrils. These data suggest that amyloid deposition is preferentially induced by amyloid fibrils with the same primary sequence as the endogenous amyloid protein. We then analysed the AEF activity of synthetic peptides, synthesized corresponding to amino acids 1-15 of mouse SAA (mSAA), 2-15 of cow SAA (bSAA), 1-15 of cat SAA (cSAA), which was the same as cheetah, and the common amino acids 33-45 of these four SAA (aSAA). We found that mSAA, bSAA and cSAA formed amyloid-like fibrils in morphology and showed similar AEF properties to those of native amyloid fibrils. Although aSAA also formed highly ordered amyloid-like fibrils, it showed weaker AEF activity than the other synthetic fibrils. Our results indicate that amyloidosis is transmissible between species under certain conditions; however, the efficiency of amyloid deposition is species-specific and appears to be related to the primary amino acid sequence, especially the N-terminal segment of the amyloid protein.

Phase II trial of oral S-1 combined with gemcitabine in metastatic pancreatic cancer.

We conducted a phase II trial of gemcitabine with S-1, oral fluorouracil (5-FU) prodrug tegafur combined with two modulators, 5-chloro-2, 4-dihydroxypyridine and potassium oxonate, to evaluate the activity and toxicity of such a combination in metastatic pancreatic cancer (MPC) patients. Patients who had pathologically proven pancreatic cancer with metastatic lesions were eligible candidates for entry into the study. S-1 was given orally (30 mg m(-2)) b.i.d. for 14 consecutive days and gemcitabine (1000 mg m(-2)) was given on days 8 and 15. The cycle was repeated every 21 days. We enrolled 33 MPC patients. The median number of cycles was eight (range 1-20). Grade 3-4 toxicities were leucopenia (33%), neutropenia (55%), anaemia (9%), thrombocytopenia (15%), anorexia (6%), fever (9%), and interstitial pneumonia (6%). Objective responses were obtained in 16 patients (one complete response and 15 partial responses; response rate, 48%; 95% confidence interval (CI), 33-65). Median survival and 1-year survival rate were 12.5 months (95% CI, 5.9-19.1) and 54% (95% CI, 36-72), respectively. Combination chemotherapy with GEM and S-1 was well tolerated and yielded a significantly high response rate.

Celecoxib upregulates endoplasmic reticulum chaperones that inhibit celecoxib-induced apoptosis in human gastric cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in cancer cells and this effect is involved in their antitumor activity. We recently demonstrated that NSAIDs upregulate GRP78, an endoplasmic reticulum (ER) chaperone, in gastric mucosal cells in primary culture. In the present study, induction of ER chaperones by NSAIDs and the effect of those chaperones on NSAID-induced apoptosis were examined in human gastric carcinoma cells. Celecoxib, an NSAID, upregulated ER chaperones (GRP78 and its cochaperones ERdj3 and ERdj4) but also C/EBP homologous transcription factor (CHOP), a transcription factor involved in apoptosis. Celecoxib also upregulated GRP78 in xenograft tumors, accompanying with the suppression of tumor growth in nude mice. Celecoxib caused phosphorylation of eukaryotic translation initiation factor 2 kinase (PERK) and eukaryotic initiation factor-2alpha (eIF2alpha) and production of activating transcription factor (ATF)4 mRNA. Suppression of ATF4 expression by small interfering RNA (siRNA) partially inhibited the celecoxib-dependent upregulation of GRP78. Celecoxib increased the intracellular Ca2+ concentration, while 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid, an intracellular Ca2+ chelator, inhibited the upregulation of GRP78 and ATF4. These results suggest that the Ca2+-dependent activation of the PERK-eIF2alpha-ATF4 pathway is involved in the upregulation of ER chaperones by celecoxib. Overexpression of GRP78 partially suppressed the apoptosis and induction of CHOP in the presence of celecoxib and this suppression was stimulated by coexpression of either ERdj3 or ERdj4. On the other hand, suppression of GRP78 expression by siRNA drastically stimulated cellular apoptosis and production of CHOP in the presence of celecoxib. These results show that upregulation of ER chaperones by celecoxib protects cancer cells from celecoxib-induced apoptosis, thus may decrease the potential antitumor activity of celecoxib.

Frequency of early and late-onset dementias in a Japanese memory disorders clinic.

The aim of this study was to compare the diagnostic profiles of patients with early (age<65 years) and late (age>or=65 years) onset of dementia in a memory disorders clinic in Japan. A total of 512 consecutive memory clinic patients were evaluated using clinical information and results of examinations. Diagnosis of dementia was made according to DSM-III-R, and that of subtypes according to standard diagnostic criteria. A total of 464 patients met the criteria for dementia. Amongst late-onset patients (n=430), Alzheimer's disease (AD) (48.1%) was the most frequent cause of dementia, followed by AD with cerebrovascular disease (CVD) (31.4%), vascular dementia (VaD) (9.1%), dementia with Lewy bodies (DLB) (3.7%), frontotemporal lobar degeneration (FTLD) (1.6%), and others (5.8%). On the contrary, amongst early onset patients (n=34), the most common dementia diagnosis was AD (38.2%), followed by VaD (23.5%), FTLD (14.7%), AD with CVD (5.9%), DLB (2.9%), and others (17.6%). FTLD and VaD were significantly more common in the early onset group. All patients, but one, with DLB and Parkinson's disease dementia were late-onset. The relative frequencies of AD, VaD, and DLB in our series are consistent with epidemiologic findings in several Western countries; however, the frequency of FTLD is not consistent with the previous findings presenting high frequency in late-onset patients in some Western countries.

Comparison of dermoscopic and histopathological findings in a mucous melanoma of the lip.

Mucous melanomas on the lip are very rare. None of the few available reports has provided a comparison of dermoscopic and histopathological findings. We describe a 33-year-old Japanese man with a mucous melanoma on the lower lip and present a comparison of our dermoscopic and histopathological findings.

Phase I trial of oral S-1 combined with gemcitabine in metastatic pancreatic cancer.

The objective of this study was to determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of S-1, an oral fluorouracil derivative, combined with gemcitabine, the current standard treatment for advanced pancreatic cancer (APC). The subjects were histopathologically proven APC patients with distant metastasis. S-1 was administered orally twice daily each day for 14 days and gemcitabine on days 8 and 15 of each cycle, and this was repeated every 21 days. Doses of each drug were planned as follows: level 1: 800/60, level 2a: 800/80, level 2b: 1000/60, level 3: 1000/80 (gemcitabine (mg m(-2))/S-1 (mg m(-2) day(-1))). In all, 21 patients with APC were enrolled. The main grade 3-4 toxicities observed during first cycle were neutropenia (33%), anaemia (10%), thrombocytopenia (14%) and anorexia (10%). There were no DLT observed in level 1. Three of six patients in level 2a had DLT and this level was considered the MTD. In all, 12 patients in level 2b had no DLT and this level was selected as the recommended dose. Applicable responses were one complete response and nine partial responses (48%). As toxicities were well tolerated and antitumour activities seem to be promising, this combination can be recommended for further phase II studies with APC.

Selective monomorphic and polymorphic HLA class I antigenic determinant loss in surgically removed melanoma lesions.

The analysis of human leukocyte antigen (HLA) class I allospecificity expression in malignant lesions has been hampered by the limited availability of HLA class I allospecificity-specific monoclonal antibodies (mAbs) which stain tissues in immunohistochemical (IHC) reactions. During the 12th International Histocompatibility Workshop, the HLA and cancer component made available a panel of mAbs capable of detecting monomorphic, locus- and allo-specific HLA class I antigenic determinants in surgically removed frozen tissue sections by IHC staining. In the present study, we have utilized this panel of mAbs to analyze the expression of HLA class I allospecificities in 33 primary and in 11 metastatic lesions surgically removed from HLA-typed patients with malignant melanoma, as this information contributes to determine the extent of HLA class I antigen abnormalities in melanoma lesions. HLA class I antigens were downregulated in six (18.2%) of the primary lesions and in six (54.5%) of the metastatic lesions. Selective loss of HLA-A and HLA-B antigens was detected in two (6.1%) and in one (3.0%), respectively, of the primary lesions, but in none of the metastases. HLA-A and HLA-B antigens were downregulated in three (9.1%) and four (36.4%) of the primary and metastatic lesions, respectively. Selective loss of one or more HLA class I allospecificities was found in 10 (33.0%) and two (18.0%) of the 33 primary and 11 metastatic melanoma lesions analyzed, respectively. HLA class I antigen abnormalities were present in 16 (48.5%) of the 33 primary lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, two lesions demonstrating selective abnormal reactivity with HLA-B locus-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A and HLA-B locus-specific mAbs, and seven lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). Furthermore, HLA class I antigen abnormalities were present in nine (81.8%) of the 11 metastatic lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A locus-specific mAb, and two lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). It cannot be ruled out that the frequency of HLA class I allospecificity abnormalities is higher, as the expression of several HLA class I allospecificities could not be investigated because of the lack of appropriate probes. The frequency of HLA class I antigen defects in primary lesions was significantly correlated with primary lesion thickness, an important prognostic marker in melanoma, arguing for a potential clinical significance of HLA class I antigen abnormalities in melanoma. In conclusion, the results of the present study (i) demonstrate that the frequency of HLA class I allospecificity abnormalities in primary melanoma lesions is markedly higher than that of total HLA class I antigen downregulation described in the literature; (ii) corroborate our previous findings that staining of melanoma lesions with mAb to monomorphic determinants of HLA class I antigens does not detect selective HLA class I allospecificity loss; and (iii) demonstrate for the first time selective loss of antigenic determinants expressed on HLA class I molecules in melanoma lesions. The latter finding indicates that at least two mAbs recognizing distinct antigenic determinants on the HLA molecule being investigated should be used for IHC staining of tissue sections in order to prove that lack of immunostaining reflects actual loss of the corresponding HLA molecule and not selective loss of antigenic determinants.

Protein composition of human metaphase chromosomes analyzed by two-dimensional electrophoreses.

A large amount of metaphase chromosomes were isolated from synchronized human cell lines by a polyamine procedure. All the chromosomal proteins extracted by an acetic acid extraction method were fully dissolved into the sample solutions for isoelectric focusing (IEF) or radical free and highly reduced (RFHR) two-dimensional electrophoreses (2-DEs). As a result, well-separated and highly reproducible 2-DE patterns were obtained. This could not be attained by an ordinary acetone precipitation method. The 2-DE patterns visualized using Coomassie Brilliant Blue (CBB) staining indicated that more than one hundred proteins were involved in the isolated metaphase chromosomes, although the most abundant proteins, histones, occupied a greater part of the chromosomal proteins. It was also shown that colcemid treatment for cell cycle synchronization had little effect on the 2-DE pattern compared to that obtained without the treatment. Furthermore, no significant differences were observed in the 2-DE patterns among the chromosomal proteins prepared from two different human cell lines, BALL-1 and K562. However, 2-DE analysis of isolated metaphase chromosomes from HeLa cells apparently showed a smaller number of proteins than the BALL-1 and K562 cell lines at a neutral pI range. The present study paves the way for elucidating protein composition of human metaphase chromosomes.