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T Monden

Publications and source records attributed to T Monden.

At least 19 recordsLinked to original sources
PubMed2000

Expression of thyrotropin-releasing hormone (TRH) receptor subtype 1 in mouse pancreatic islets and HIT-T15, an insulin-secreting clonal beta cell line.

Thyrotropin-releasing hormone (TRH), originally isolated as a hypothalamic hormone, has been reported to be present and released from the pancreatic beta cells, affecting pancreatic functions. However, it still remains unclear whether TRH receptor is expressed in the pancreas. In the present study, we characterized TRH receptors (TRHR) in mouse pancreatic islets and HIT-T15 cells, a hamster clonal beta cell line. RT-PCR study showed significant expression of TRHR subtype 1 (TRHR1) mRNA in both mouse pancreatic islets and HIT-T15 (HIT) cells. In contrast, there was no expression of TRHR2 mRNA, a novel subtype of TRHR which is expressed predominantly in the central nervous system. Sequencing analysis demonstrated that TRHR1 of the islets was identical to that in the pituitary, and cloned hamster TRHR1 shared 93.3 % homology with that of the mouse at the nucleic acid level. Northern blot analysis of TRHR 1 mRNA in HIT-T15 cells showed a single strong hybridization signal approximately 3.7 kb in length. Furthermore, Scatchard plot analysis in HIT-T15 cells revealed that the Kd value for MeTRH was 0.63 nM. Significant elevation of intracellular calcium concentration was observed in response to as little as 10 nM TRH , and this was not affected by removal of extracellular calcium. This is the first description indicating the presence of functional TRH receptor subtype 1 in the pancreatic beta cells, and our observations suggested the regulation of pancreatic function by TRH through autocrine or paracrine mechanisms.

Amino Acid Sequence↗
PubMed2000

Selective augmentations of intratumoral 5-fluorouracil concentration by local immunotherapy with OK-432 and fibrinogen.

PURPOSE: Pyrimidine nucleoside phosphorylase is an enzyme that converts 5'-deoxy-5-fluorouridine into its active metabolite, 5-fluorouracil. In colorectal cancer tissue pyrimidine nucleoside phosphorylase has been proven to be produced by macrophages in the cancer stroma despite presence of the cancer cells. We reported that local immunotherapy with OK-432 and fibrinogen induced aggregation of macrophages in the cancer stroma and enforced their pyrimidine nucleoside phosphorylase expression. Thus it was hypothesized that if colon cancer were treated with 5'-deoxy-5-fluorouridine, the 5-fluorouracil concentration in cancer tissues would be enhanced by local immunotherapy. The present study was conducted to investigate whether local immunotherapy for colon cancer could increase the intratumoral 5-fluorouracil concentration in patients given chemotherapy with 5'-deoxy-5-fluorouridine. METHODS: Twenty patients with resectable colorectal cancer were examined in this study. They were given 5'-deoxy-5-fluorouridine (600 mg/day) orally for seven days preoperatively. Nine randomly selected patients underwent intratumoral injection of OK-432 mixed with fibrinogen, which was performed on the third preoperative day (OK-432 and fibrinogen plus 5'-deoxy-5-fluorouridine group); eleven patients were given oral 5'-deoxy-5-fluorouridine only (5'deoxy-5-fluorouridine group). The 5-fluorouracil concentration in tumor tissue and normal colon mucosa tissue was measured, and the influence of the local immunotherapy was assessed. RESULTS: The 5-fluorouracil concentration in the cancer tissue was increased by the local immunotherapy, whereas that in the normal colon mucosa was not influenced. Thus, the influence of local immunotherapy was selective to the cancer tissue where the mixture of OK-432 and fibrinogen was injected. CONCLUSION: In patients with colorectal cancer, selective high 5-fluorouracil concentration in the cancer tissue could be achieved by a combination of 5'-deoxy-5-fluorouridine and local immunotherapy with a mixture of OK-432 and fibrinogen.

Aged↗
PubMed2000

Expression of tumor suppressor gene p16(INK4) products in primary gastric cancer.

Recent studies have shown that the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) represents an indicator for patients' outcome in several human malignancies including gastric cancer. However, the clinicopathologic value of another class of CDK inhibitor, p16(INK4), has not been determined. In a retrospective study, we examined the expression of p16(INK4) by immunohistochemical assay of 80 samples of primary gastric cancers and their adjacent nonneoplastic mucosas. Less than 10% of non-tumor gastric mucosal cells were p16(INK4) positive, whereas the expression of p16(INK4) in gastric cancer cells varied widely from 0 to 100% (mean, 24.5%). The expression of p16(INK4) was not seen in 11.3% (9/80) of the cancer cases, but in 65% (52/80) this protein was even overexpressed when compared with the nonneoplastic mucosa. A clinicopathologic survey indicated that a low or no expression of p16(INK4) was associated with poorly differentiated carcinoma (p = 0.0133), but the level of expression did not correlate with other parameters including patients' prognosis or with the expression of the pRb protein. In an effort to explore the underlying mechanism for the p16(INK4)-negative cases, a prospective study was also performed on 20 cases of gastric cancer to compare the level of the p16(INK4) protein with the methylation status of the p16(INK4) promoter. Gastric cancer tissues with methylation expressed significantly lower levels of the p16(INK4) protein (p = 0.0013) and two of them lacked p16(INK4) expression altogether, whereas all the cancer tissues without methylation expressed it. These findings suggest that the p16(INK4) protein may be associated with differentiation of gastric cancer tissues and that methylation of the p16(INK4) promoter may, in part, account for the loss of p16(INK4) expression.

Blotting, Western↗
PubMed2000

Requirement of thyrotropin-releasing hormone for the postnatal functions of pituitary thyrotrophs: ontogeny study of congenital tertiary hypothyroidism in mice.

We recently reported that TRH-deficient mice showed characteristic tertiary hypothyroidism. In the present study, we investigated how this tertiary hypothyroidism occurred particularly in pre- and postnatal stages. Immunohistochemical analysis revealed a number of TSH-immunopositive cells in the TRH-/- pituitary on embryonic day 17.5 and at birth. The mutant pituitary at birth in pups born from TRH-deficient dams also showed no apparent morphological changes, indicating no requirement of either maternal or embryonic TRH for the development of pituitary thyrotrophs. In contrast, apparent decreases in number and level of staining of TSH-immunopositive cells were observed after postnatal day 10 in mutant pituitary. Similar decreases were observed in the 8-week-old mutant pituitary, while no apparent changes were observed in other pituitary hormone-producing cells, and prolonged TRH administration completely reversed this effect. Consistent with these morphological results, TRH-/- mice showed normal thyroid hormone levels at birth, but the subsequent postnatal increase was depressed, resulting in hypothyroidism. As expected, TSH content in the TRH-/- pituitary showed a marked reduction to only 40% of that in the wild type. Despite hypothyroidism in the mutant mice, both the pituitary TSHbeta and alpha mRNA levels were lower than those of the wild-type pituitary. These phenotypic changes were specific to the pituitary thyrotrophs. These findings indicated that 1) TRH is essential only for the postnatal maintenance of the normal function of pituitary thyrotrophs, including the normal feedback regulation of the TSH gene by thyroid hormone; 2) neither maternal nor embryonic TRH is required for normal development of the fetal pituitary thyrotroph; and 3) TRH-deficient mice do not exhibit hypothyroidism at birth. Moreover, reflecting its name, TRH has more critical effects on the pituitary thyrotrophs than on other pituitary hormone-producing cells.

Animals↗
PubMed1999

DNA binding and interaction with the nuclear receptor corepressor of thyroid hormone receptor are required for ligand-independent stimulation of the mouse preprothyrotropin-releasing hormone gene.

A negative thyroid hormone response element (TRE) in the mouse preprothyrotropin-releasing hormone (TRH) gene was previously mapped within the proximal promoter element between -83 and +53 that contained a TRE half-site motif at -57 (-57TGACCT-51). In transfection experiments, the promoter activity is stimulated by unliganded thyroid hormone receptor (TR) and T3 reverses the basal promoter stimulation. In this study, we determined whether the direct binding of TR to the TRE half-site in the mouse TRH gene is required for the ligand-independent stimulation using a transient transfection assay into CV-1 cells and electrophoretic mobility shift assays (EMSA). In addition, the role of a corepressor protein for the ligand-independent stimulation was examined using a putative splicing variant of the nuclear receptor corepressor (N-CoRI). Point mutations introduced into the TRE half-site at -57 eliminated the binding of TR and the stimulatory effect of unliganded TR. Two mutant TRs lacking DNA-binding activity and two CoR box mutant TRs showed no stimulation in the wild-type TRH promoter. The cotransfected N-CoRI potentiated the ligand-independent stimulation by the wild-type TR, but did not compensate for the impaired function of the CoR box mutant TR. In EMSA, TR strongly bound as homodimers and weakly as heterodimers with retinoid X receptor (RXR) to the element containing the TRE half-site at -57. Binding of TR to the TRE half-site was essential to form homo- and heterodimers, and the RXR binding site appeared to be located downstream of the TRE half-site. In vitro translated N-CoRI preferentially bound TR homodimers over TR/RXR heterodimers. These results collectively suggest that the DNA-bound TR/corepressor complex might be directly involved in the ligand-independent stimulation of the mouse TRH gene promoter.

Animals↗
PubMed1999

Molecular characteristics of poorly differentiated adenocarcinoma and signet-ring-cell carcinoma of colorectum.

In a series of 45 poorly differentiated adenocarcinomas (por) and 7 signet-ring-cell carcinomas (sig) of the colorectum, K-ras gene mutation, p53 immunostaining and microsatellite instability (MSI) were analyzed for a comparison with 46 cases of colorectal carcinomas of the well or moderately differentiated type (well/mod). In addition, the mutations of simple repeated sequences in the transforming-growth-factor-beta type-II receptor (T beta R-II) gene and the BAX gene were analyzed as possible targets for DNA replication errors. Mutation of the K-ras gene in the por, sig and well/mod specimens was detected in, respectively, 22%, 11% and 48%, positive immunostaining for p53 in 41.8%, 28.6% and 60.3%, and MSI in 36%, 30% and 4%. Frameshift mutation of the T beta R-II gene was detected in 27.5% of the por and none of the sig specimens, while corresponding figures for mutation of the BAX gene were 15.7% and 0%. Significant differences between the por and well/mod tumors were found in the occurrence of K-ras mutation at codons 12 and 13, and MSI. Clinicopathologically, the tumor status of por with MSI was found to significantly correlate with the tumor's location in the proximal colon. In cases without MSI and sig, no frameshift mutation of either the T beta R-II or the BAX gene was found. These results suggest that poorly differentiated and signet-ring-cell carcinomas have a genetic background different from that of well or moderately differentiated carcinomas of the colorectum, and that DNA-replication error is at least partly involved in the carcinogenesis of these specific types of colorectal carcinomas.

Adenocarcinoma↗
PubMed1999

Laparoscopic surgery for blind pouch syndrome following Roux-en Y gastrojejunostomy: report of a case.

We report herein the case of a 59-year-old man in whom blind pouch syndrome was successfully treated by laparoscopic surgery. The patient had undergone distal gastrectomy and Roux-en Y gastrojejunostomy for a peptic ulcer 35 years previously, and had been suffering from watery diarrhea, anemia, weight loss, and pain in the left upper quadrant of his abdomen for several years. Long-term insufficient oral intake and the malabsorption of nutrients had resulted in severe emaciation. Gastrointestinal contrast study revealed a large blind pouch, 30 x 23cm in diameter, draining into the gastrojejunostomy. Laparoscopic resection of the blind pouch was performed. Despite the presence of dense intraabdominal adhesions, we identified the blind pouch with the help of tattoo marks that had been made at the neck of the pouch preoperatively. After thoroughly dissecting the adhesions around the pouch, we resected the pouch at the neck. The patient had an uneventful postoperative course. This case report demonstrates that large blind pouches such as this may be effectively treated using laparoscopic surgery.

Anastomosis, Roux-en-Y↗
PubMed1999

Abundance of cyclo (His-Pro)-like immunoreactivity in the brain of TRH-deficient mice.

Cyclo(His-Pro) or CHP was initially discovered as a metabolite of thyrotropin-releasing hormone (TRH) resulting from the action of the enzyme Pyroglutamyl aminopeptidase. Physiologic and pharmacologic studies that followed this initial discovery provided indirect evidence that all CHP may not be derived from TRH. However, the recent availability of a TRH-deficient mouse has made it possible to reinvestigate whether CHP is derived from TRH. In the present study, we examined distribution of CHP and TRH in TRH-deficient mice. Northern blot analysis confirmed the absence of preproTRH mRNA in both the hypothalamus and the cortex of TRH-deficient mice. Brains from the wild-type and TRH-deficient mice were dissected into 7 regions, and TRH and CHP concentrations were determined by specific radioimmunoassay (RIA) in each region. Whereas TRH was identified in all regions of the wild-type brain, with the highest concentration in the hypothalamus, no detectable TRH was observed in any region in the TRH-deficient mice. While CHP-like immunoreactivity (CHP-LI) was present in all regions in the wild-type brain, its concentration was reduced by approximately 50% in the hypothalamus and cerebral cortex of TRH-deficient mice, with no change in other brain regions. Furthermore, the CHP-LI present in the brain of TRH-deficient mice was immunologically and chromatographically identical to synthetic CHP. These findings strongly suggest that a portion of the CHP in the brain is derived from sources other than TRH.

Animals↗
PubMed1999

Regulation of the mouse preprothyrotropin-releasing hormone gene by retinoic acid receptor.

Retinoic acid (RA) has been reported to inhibit the secretion and synthesis of the pituitary TSH in vivo and in vitro. However, little is known about the influence of RA on the expression of the prepro-TRH gene. We therefore investigated whether the promoter activity of the mouse TRH gene is directly regulated by RA using a transient transfection assay into CV-1 cells. In the absence of cotransfected RA receptor (RAR), all-trans-RA did not affect the promoter activity. In contrast, the cotransfected RARalpha significantly stimulated promoter activity in the absence of ligand, and all-trans-RA reversed basal promoter activation. The cotransfected thyroid hormone receptor-beta (TRbeta), but not 9-cis-RA receptor (RXR), had an additive effect on the RAR-dependent stimulation. TR and RAR can similarly interact with the corepressor proteins, and the cotransfected nuclear receptor corepressor (N-CoR) has been demonstrated to augment the transcriptional stimulation of the TRH gene by unliganded TR. As observed with TR, the coexpression of a N-CoR variant significantly enhanced the ligand-independent stimulation by RAR. A mutant RAR (RAR403) lacking the C-terminal activation function-2 (AF-2) activation domain that was essential for ligand-induced corepressor release constitutively stimulated the promoter activity. The constitutive stimulation by RAR403 was augmented by the cotransfected N-CoR variant. A deletion analysis of the 5'-flanking region of the TRH gene revealed that the minimal promoter region for the regulation by RAR was -83 to +53, with a consensus half-site motif for the thyroid hormone response element at -57. In contrast to the strong binding of TR to the thyroid hormone response element half-site in gel retardation assays, no binding of RAR homodimer, RAR/ RXR heterodimer, or RAR/TR heterodimer was observed to the minimal promoter region. These results collectively suggest that RAR without heterodimerization with RXR and TR regulates transcription of the mouse TRH gene in cooperation with the corepressor, and that the DNA binding of RAR appeared to be unnecessary for regulation of the TRH gene promoter.

Animals↗
PubMed1999

p120 acts as a specific coactivator for 9-cis-retinoic acid receptor (RXR) on peroxisome proliferator-activated receptor-gamma/RXR heterodimers.

p120 was originally isolated as a novel nuclear co-activator for thyroid hormone receptor. In this study, we characterized its interaction and transactivation of peroxisome proliferator-activated receptor-gamma (PPARgamma) and 9-cis-retinoic acid receptor (RXR) heterodimers. Transient transfection study revealed that p120 enhanced the transcriptional activation of PPARgamma/RXR induced by PPARgamma- or RXR-specific ligands. In the glutathione-S-transferase pull-down assay, while steroid receptor coactivator-1 showed apparent interactions with both RXR and PPARgamma, p120 bound only to RXR in a 9-cis-retinoic acid (RA)-dependent manner and also did not bind to PPARgamma even in the presence of thiazolidinediones. The yeast two-hybrid analysis showed no interaction of p120 with PPARgamma under any conditions, and electophoretic mobility shift assay showed apparent DNA-PPARgamma/RXR/p120 complex formation only in the presence of 9-cis-RA. Furthermore, the yeast three-hybrid assay clearly revealed a significant interaction between p120 and PPARgamma via RXR of PPARgamma/RXR heterodimer only in the presence of 9-cis-RA. These findings indicate that p120 acts as a specific co-activator for the RXR of PPARgamma/RXR heterodimer in a 9-cis-RA-dependent manner.

Adipose Tissue↗
PubMed1999

Human thyrotropin-releasing hormone-associated peptide 3 (hTAP-3) in serum.

Human thyrotropin-releasing hormone (TRH)-associated peptide 3 (hTAP-3), one of the cryptic peptides resulting from the proteolytic processing of preproTRH to produce TRH, was measured in human plasma from normal, hyperthyroid, and hypothyroid subjects. The dilution curve of hTAP-3 immunoreactivity in the serum paralleled the standard curve of the radioimmunoassay. HPLC analysis revealed a single strong immunoreactive peak, which corresponded to the authentic peptide, hTAP-3. The half-life of hTAP-3 in serum was approximately 3.5 min, and the addition of aprotinin and EDTA completely prevented its degradation. In hyperthyroid patients, plasma concentrations of hTAP-3 were significantly higher than those in the control group and hypothyroid patients, but no correlation was found between its level and that of thyroid hormone. These findings indicate the existence of intact hTAP-3 in the human serum and increases in plasma hTAP-3 levels in hyperthyroid patients, suggesting that blood hTAP-3 may be derived from the peripheral organs rather than the hypothalamus.

Adult↗
PubMed1999

Paradoxical increase in retinoblastoma protein in colorectal carcinomas may protect cells from apoptosis.

The retinoblastoma (Rb) gene is inactivated in a variety of human cancers, but in colorectal carcinomas there is frequently increased expression of this gene. This is paradoxical in view of the known role of Rb as a tumor suppressor gene. In the present study, we compared the levels of expression of the Rb protein (pRb) in normal human colorectal mucosa, adenomatous polyps, and carcinomas by immunohistochemistry. In vitro studies were also done to examine the phenotypic effects of an antisense oligodeoxynucleotide (AS-Rb) targeted to Rb mRNA in the HCT116 colon carcinoma cell line that expresses a relatively high level of pRb. The incidence of pRb-positive cells was increased during multistage colorectal carcinogenesis. In vitro treatment of HCT116 cells with AS-Rb decreased the level of pRb by about 70% and also decreased the levels of the cyclin D1 protein and cyclin D1-associated kinase activity. AS-Rb inhibited growth of HCT116 cells and induced apoptosis. Reporter assays indicated about a 17-fold increase in E2F activity. These findings suggest that the increased expression of pRb in colorectal carcinoma cells may provide a homeostatic mechanism that protects them from growth inhibition and apoptosis, perhaps by counterbalancing potentially toxic effects of excessive E2F activity.

Apoptosis↗
PubMed1998

Genomic organization and promoter function of the mouse uncoupling protein 2 (UCP2) gene.

We cloned and characterized the mouse uncoupling protein 2 (UCP2) gene and its promoter region. The gene spans approximately 6.3 kb and contains eight exons and seven introns. Two short exons are located in the 5' untranslated region, and each of the remaining exons encodes one of the transmembrane domains. 3'-RACE analysis showed that a polyadenylation signal 257 bp downstream from the stop codon was functional. Primer extension analysis indicated a single transcriptional start site 369 bp upstream from the translational start site. The promoter region lacks both TATA and CAAT boxes but is GC-rich. A construct containing 1250 bp of the promoter region showed significant activity in all 6 cell lines examined, and the region between -160 and -678 bp exhibited strong positive regulatory activity. These features of the UCP2 gene are different from those of the UCP1 gene and may contribute to its ubiquitous expression.

Amino Acid Sequence↗
PubMed1998

Identification of a novel gene marker specific for epithelial cells by utilizing a 3'-directed cDNA library.

To achieve reliability of molecular diagnosis using reverse transcription-PCR (RT-PCR), we established a unique method to search for a novel gene marker specific for colonic epithelial cells. Of eight candidate genes selected from a 3'-directed cDNA library in colonic mucosa, two genes were expressed in normal mucosa and cancer of the colon but not in either normal lymph node or normal liver tissue. Known sequences of these genes were reported to be located in the 3' noncoding region, and an additional sequence just upstream to gs04094 (one of the candidate genes) was determined. According to the newly identified sequence, we designed a new set of primers so that we could distinguish the DNA fragment amplified in RT-PCR from that in genomic PCR. RT-PCR using these primers demonstrated that gs04094 was expressed in all of 10 primary colon cancers and 4 liver metastases from colon cancer but in none of 5 normal lymph nodes, 10 peripheral blood samples, and 2 normal liver tissues. Sensitivity of this method was so high as to detect gs04094 mRNA in 10(-6) microg of colon cancer RNA per 1 microg of normal lymph node RNA. Thus, our strategy to search for a novel gene marker using 3'-directed cDNA library proved to be highly efficient.

Animals↗
PubMed1998

PyNPase expression in human colon cancer.

Although colorectal cancer tissue is rich in pyrimidine nucleoside phosphorylase (PyNPase), there is no consensus as to whether cancer cells or stromal cells predominately express PyNPase. We micro-dissected OCT compound embedded frozen tissue sections into epithelial and stromal components and then analyzed the extracted samples separately. The PyNPase expression level was higher in stromal cells than in cancer cells and the difference increased with inflammation induced by the immunostimulator OK432. These results suggest that stromal cells are the major PyNPase source in colorectal cancer.

Adult↗
PubMed1998

Localization and expression of p27KIP1 in multistage colorectal carcinogenesis.

The cyclin-dependent kinase inhibitor p27Kip1 can inhibit the G1 to S transition of the cell cycle and is a putative tumor suppressor. However, our laboratory found that a variety of human cancer cell lines express relatively high levels of this protein and that this is often associated with increased expression of cyclin D1 or cyclin E. Therefore, in the present study we analyzed by immunohistochemistry the expression of p27Kip1 in a series of human tissue samples representing various stages of colon carcinogenesis, using 20 samples of normal colon mucosa, 20 hyperplastic polyps, 19 samples of adenomatous polyps, and 40 samples of various types of colorectal carcinomas. Parallel immunostaining was done for cyclin D1 and also for Ki67 to evaluate cell proliferation. An additional 17 human colon carcinoma samples, together with paired adjacent normal mucosa samples, were analyzed for levels of expression of the p27Kip1 protein by Western blot analysis, and 7 of these pairs of samples were examined by Northern blot analysis for levels of p27Kip1 mRNA. We did not find a positive or negative correlation between p27Kip1 expression and cell proliferation in the normal mucosa and tumor samples. There was, however, an inverse correlation between p27Kip1 and Ki67 expression in the lymphoid follicles present in the colonic mucosa. There was no evidence for a consistent increase or decrease in p27Kip1 expression in the mucosal cells during colon carcinogenesis, because the mean values for percentage p27Kip1-positive cells were similar in the normal mucosa, adenomatous polyps, and carcinoma samples. This is in contrast to Ki67 and cyclin D1 expression, which did show significant increases in mean values with tumor development. A subset (35%) of the carcinomas displayed diffuse cytoplasmic staining, in addition to nuclear staining, for p27Kip1, and in these cases the percentage of cells that were positive for p27Kip1 was higher than in cases that had only nuclear staining. There was a significant correlation between p27Kip1 expression and tumor grade; ie., well and moderately differentiated carcinomas had high p27Kip1 expression, whereas poorly differentiated carcinomas had lower expression. The Western blot analysis data on p27Kip1 expression confirmed this correlation. Comparisons of Northern and Western blots did not show a correlation between the level of p27Kip1 mRNA and the corresponding protein, a finding consistent with evidence that the p27Kip1 protein is regulated mainly via a posttranscriptional mechanism. The immunostaining studies revealed a significant correlation between high p27Kip1 protein expression and high cyclin D1 expression in the adenomatous polyps and in the subset of carcinomas that had only nuclear p27Kip1 expression. This may reflect the existence of a homeostatic feedback mechanism that is lost in the high-grade carcinomas that express low levels of p27Kip1.

Adult↗
PubMed1998

Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections.

We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-micron-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as beta-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.

Adenoma↗