In human skin, specific keratin markers reflect on normal differentiation and pathologic conditions. This experiment focused on the expressional pattern of keratin 10 (K10: normal differentiation marker), and keratin 8 & 13 (K8 & K13: pathologic differentiation marker) together with their cellular localization after treating HaCaT cells with 12-Otetradecanoylphorbol 13-acetate (TPA). The cells were treated with TPA at 0, 0.1, 1 microgram/ml for 2 hours or 6 hours. Morphologic studies revealed that TPA treatment changed the shape of cells into the fibroblast-like cells with highly folded nuclear membrane and reduced number of the desmosome. The results of indirect immunofluorescent staining and Northern blotting analysis showed that TPA considerably down-regulated the expression of K10, while markedly up-regulating the expression of K8 and K13 both at protein and mRNA levels. Furthermore, by simultaneous staining for keratins and DNA content in flow cytometry, it was found that TPA increased the expression of K8 and K13 dramatically at the S-G2-M phase of the cells. In conclusion, these changes induced by TPA in HaCaT cells may indicate a close relationship between the morphologic change and the altered expression of keratin subfamilies. It also suggests that TPA known as a tumor promotor may directly induce the potentially malignant cells even without the support of tumor initiator.
Blotting, Northern ; Desmosomes ; DNA ; Flow Cytometry ; Humans ; Keratin-10 ; Keratin-8 ; Nuclear Envelope ; RNA, Messenger ; Skin

PURPOSE: the most important biological behavior of breast cancer is its invasive potential and many efforts was made to reveal the factors related with the invasiveness of breast cancer cells. Some researchers reported that intermediate filament biology could represent an emerging and exciting field in tumor biology with respect to tumor aggressiveness and invasiveness. There are some experimental evidences that co-expression of vimentin, a interfilament marker indicative of mesenchymal lineage, and cytokeratin interfilaments can be correlated with invasiveness and metastatic deposits. So, we tried to determine the role of intermediate filaments such as cytokeratins and vimentin with respect of bone marrow micrometastases. METHODS: Expression of cytokeratins 8, 18, 19 and viementin were immunohistochemically evaluated. Detection of bone marrow micrometastases was preformed through RT-PCR targeting mRNA of cytokeratin 19. In order to compare the study group by the expression extent of cytokeratins, the case expressing 50% or more of observed cells was classified into the case with high expression and the case expressing 49% or less was classified into the case with low expression. RESULTS: The only cytokeratin of high expression representing the risk of bone marrow micrometastases was cytokeratin 8. Vimentin expression by itself did not show any significance indicating bone marrow micrometastases. However, The cases possesing cytokeratin 8, 18, and 19 expression, altogether 75% or more showed a significantly high risk to bone marrow micrometastases. In that cases, addition of vimentin expression allowed a more higher possibility of bone marrow micrometastases. CONCLUSION: A high expression of cytokeratin 8 among cytokeratins was related with bone marrow metastases. However, vimentin expression by itself did not show any realtionship with bone marrow metastases. So, a further study is needed in order to reveal the role of vimentin expression in progression and metastases of breast cancer.
Biology ; Bone Marrow ; Breast Neoplasms* ; Breast* ; Intermediate Filaments ; Keratin-19 ; Keratin-8 ; Keratins ; Neoplasm Metastasis* ; Neoplasm Micrometastasis ; RNA, Messenger ; Vimentin*

Sphingosylphosphorylcholine (SPC) is significantly increased in the malicious ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments in PANC-1 cells. The reorganization contributes to the viscoelasticity of metastatic cancer cells resulting in increased migration. Recently, we reported that transglutaminase-2 (Tgase-2) is involved in SPC-induced K8 phosphorylation and reorganization. However, effects of Tgase-2 inhibitors on SPC-induced K8 phosphorylation and reorganization were not clearly studied. We found that ethacrynic acid (ECA) concentration-dependently inhibited Tgase-2. Therefore, we examined the effects of ECA on SPC-induced K8 phosphorylation and reorganization. ECA concentration-dependently suppressed the SPC-induced phosphorylation and perinuclear reorganization of K8. ECA also suppressed the SPC-induced migration and invasion. SPC induced JNK activation through Tgase-2 expression and ECA suppressed the activation and expression of JNK in PANC-1 cells. These results suggested that ECA might be useful to control Tgase-2 dependent metastasis of cancer cells such as pancreatic cancer and lung cancers.
Ascites ; Ethacrynic Acid* ; Humans ; Keratin-8* ; Lung Neoplasms ; Neoplasm Metastasis ; Pancreatic Neoplasms ; Phosphorylation*

PURPOSE: Whereas lymph node metastasis in gastric cancer is an important prognostic factor, the prognostic relevance of occult tumor cells in lymph nodes has not yet been elucidated. The aims of this study were to assess the incidence of micrometastases of lymph nodes in patients with submucosally invaded gastric cancer and to investigate whether micrometastases of lymph nodes have prognostic significance. METHODS: In order to evaluate the incidence of lymph node micrometastases in patients with submucosal gastric cancer, 1423 lymph nodes taken from 55 patients were assessed by immunohistochemical technique using a monoclonal anti-human cytokeratin-8. Clinicopathologic parameters and prognoses were compared between patients with and without micrometastases. RESULTS: The incidence of nodal involvement by tumor cells in 55 patients with submucosal gastric cancer increased from 20.0% (11 patients) by hematoxylin-eosin (H-E) staining to 30.8% (17 patients) by immunohistochemical staining. Nodal positivity in this study increased from 0.8% (12/1423 nodes) by H-E staining to 3.2% (45/1423 nodes) by immunohistochemical staining (p=0.003). The presence of cytokeratin positivity was not related to various clinicopathologic factors. As estimated by the Kaplan-Meier lifetable methods, there was no significant difference in the five-year survival rate between the micrometastases negative and positive groups (94.8% and 94.1%, respectively). CONCLUSION: The presence of nodal micrometastases detected by immunohistochemical technique is an interesting phenomenon, however clinically it seems to be of only weak prognostic value in submucosal gastric cancer.
Humans ; Incidence* ; Keratin-8 ; Keratins ; Lymph Nodes* ; Neoplasm Metastasis ; Neoplasm Micrometastasis* ; Prognosis ; Stomach Neoplasms* ; Survival Rate

The stiffness of cancer cells is attributable to intermediate filaments such as keratin. Perinuclear reorganization via phosphorylation of specific serine residue in keratin is implicated in the deformability of metastatic cancer cells including the human pancreatic carcinoma cell line (PANC-1). 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a potent tumor promoter and protein kinase C (PKC) activator. However, its effects on phosphorylation and reorganization of keratin 8 (K8) are not well known. Therefore, we examined the underlying mechanism and effect of TPA on K8 phosphorylation and reorganization. TPA induced phosphorylation and reorganization of K8 and transglutaminase-2 (Tgase-2) expression in a time- and dose-dependent manner in PANC-1 cells. These effects peaked after 45 min and 100 nM of TPA treatment. We next investigated, using cystamine (CTM), Tgase inhibitor, and Tgase-2 gene silencing, Tgase-2's possible involvement in TPA-induced K8 phosphorylation and reorganization. We found that Tgase-2 gene silencing inhibited K8 phosphorylation and reorganization in PANC-1 cells. Tgase-2 gene silencing, we additionally discovered, suppressed TPA-induced migration of PANC-1 cells and Tgase-2 overexpression induced migration of PANC-1 cells. Overall, these results suggested that TPA induced K8 phosphorylation and reorganization via Tgase-2 expression in PANC-1 cells.
Cell Line ; Cystamine ; Gene Silencing ; Humans ; Intermediate Filaments ; Keratin-8* ; Phosphorylation* ; Protein Kinase C ; Serine

OBJECTIVETo study the mechanism of liver fibrogenesis and to find new non-invasive biomarkers.

METHODIn this study, we used subcellular proteomic technology to study the plasma membrane proteins related to immune or alcohol induced liver fibrosis. Rat liver fibrosis models were induced by pig serum or alcohol injection. The liver fibrogenesis were detected by James's staining in the rat models after 2, 4, 6 and 8 weeks of treatment. The liver plasma membrane (PM) of the 2- and 8-week treatment model rats were enriched by two-step sucrose density gradient centrifugation. The purity of PM was verified by western blotting, and the plasma membrane proteins were extracted and analyzed by 2 DE. The differentially expressed proteins were identified by LC-MS/MS. Cellular location and function of these identified differential protein were classified.

RESULTSImmune or alcohol induced liver fibrosis rat models were successfully established. Liver plasma membrane was significantly enriched after sucrose density ultracentrifugation treatment. 87 differential protein spots were find out by 2DE combined with LC-MS/MS from the liver plasma membrane proteins of the 2- and 8-week treatment rat models, which corresponded to 30 non-redundant proteins including annexin A2, keratin 8 and keratin 18.

CONCLUSIONSA list of differentially expressed proteins relate to liver fibrosis were successfully identified. Differential proteins such as annexin A2, keratin 8 and keratin 18 could be new biomarkers for liver fibrosis diagnosis.

Alcohols ; adverse effects ; Animals ; Female ; Keratin-18 ; metabolism ; Keratin-8 ; metabolism ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; chemically induced ; immunology ; metabolism ; pathology ; Male ; Proteome ; metabolism ; Rats ; Rats, Sprague-Dawley

Carcinoma, Renal Cell ; metabolism ; pathology ; surgery ; Diagnosis, Differential ; Humans ; Keratin-18 ; metabolism ; Keratin-19 ; metabolism ; Keratin-8 ; metabolism ; Kidney Neoplasms ; metabolism ; pathology ; surgery ; Male ; Middle Aged ; Nephrectomy ; Neprilysin ; metabolism ; Racemases and Epimerases ; metabolism

OBJECTIVETo construct a GFP/Puro double-labeled lentiviral expression vector for CK8 silencing and assess the effects of CK8 silencing on cell apoptosis.

METHODSThe siRNA sequences of CK8 were inserted into the lentiviral expression vector GV248 and transfected into 293T cells with the packaging plasmids PMD and SPA. The lentivirus was collected at 24 and 36 h post-transfection. Flow cytometry was used to detect the virus titer and the positive cells were selected with puromycin. The knockdown of CK8 was examined by Western blotting. The effect of CK8 down-regulation on cell apoptosis induced by cisplatin was detected with Annexin V/PI staining.

RESULTS AND CONCLUSIONWe successfully constructed CK8 interference lentiviral vector and obtained a stable cell line with CK8 knock-down that was sensitive to cisplatin-induced apoptosis.

Apoptosis ; Cell Line ; Down-Regulation ; Genetic Vectors ; Humans ; Keratin-8 ; genetics ; Lentivirus ; Plasmids ; RNA Interference ; RNA, Small Interfering ; Transfection

BACKGROUND AND OBJECTIVES: This study investigated the expression of cytokeratin 8, 18, 19 with low molecular weight, which have been classified as a group of simple epithelium-related marker for advanced squamous cell carcinoma of the larynx. MATERIALS AND METHODS: Detection of cytokeratin expression was performed by immunohistochemical study using antikeratin monoclonal antibodies (CAM5.2, RCK108). Immunohistochemical study was used further to detect the presence of p53 mutation in larynx carcinoma, and PCR was performed to detect the infection of HPV. We then tried to draw relationship among these factors with regard to advanced larynx carcinoma. RESULTS: Cytokeratin 8, 18 (CAM5.2) was detected in 17 cases among the 19 advanced larynx carcinoma, and in 3 cases among the 15 normal larynx. Cytokeratin 19(RCK108) was detected in 18 cases among the advanced larynx carcinoma, and in 11 cases among the 15 normal larynx. HPV DNA was detected in 4 of the 19 cases of larynx carcinoma. With regard to subtypes of HPV, HPV 16 was detected in 2 cases. And p53 was detected in 6 out of the 19 cases of larynx carcinoma. There was no correlation among the cytokeratin expression, the p53 expression, and the HPV infection. CONCLUSION: This results show that cytokeratin 8, 18 (CAM5.2) expression might be a meaningful parameter in malignant change of the larynx, but the prognostic role of the cytokeratin and the role of p53 and HPV in cytokeratin expression in larynx carcinoma was not confirmed.
Antibodies, Monoclonal ; Carcinoma, Squamous Cell ; DNA ; Human papillomavirus 16 ; Humans* ; Keratin-8 ; Keratins* ; Larynx ; Molecular Weight ; Papilloma* ; Polymerase Chain Reaction

OBJECTIVETo study the clinicopathologic features and differential diagnosis of proximal gastric mucosa and mucosa of Barrett's esophagus (BE) in biopsy specimens.

METHODThirty-eight cases of Barrett's esophagus (diagnosed using WHO criteria) and 44 cases of proximal gastric mucosa were studied by immunohistochemistry (for CK7, CK20, CK4, CK8, S-100 protein, MUC6, COX2 and bcl-2) and fluorescence in-situ hybridization (FISH) (for hTERC gene). The pathologic features were analyzed.

RESULTSThe differences in expression of CK7, CK20, MUC6, COX2 and bcl-2 between BE and proximal gastric mucosa with intestinal metaplasia were not statistically significant (P > 0.05). There was however a statistically significant difference in expression of S-100 protein (P < 0.05). The expression of CK7/CK4 and CK7/CK8 in BE showed positive correlation (P < 0.05). However, such correlation was not demonstrated in proximal gastric mucosa (P > 0.05). The results of hTERC gene expression by FISH showed a statistically significant difference between the two groups: 57.9% (22/38) in BE and 13.6% (6/44) in proximal gastric mucosa (P < 0.05).

CONCLUSIONSThe significance of CK7 and CK20 expression is uncertain in the differential diagnosis between BE and proximal gastric mucosa. On the other hand, positivity for CK7/CK4/CK8 may support the diagnosis of BE and play a role in distinguishing between the two groups. S-100 protein expression and detection of hTERC gene amplification also contribute to the diagnosis of BE.

Barrett Esophagus ; genetics ; metabolism ; pathology ; Gastric Mucosa ; metabolism ; pathology ; Gene Amplification ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Keratin-20 ; metabolism ; Keratin-4 ; metabolism ; Keratin-7 ; metabolism ; Keratin-8 ; metabolism ; Metaplasia ; genetics ; metabolism ; pathology ; RNA ; genetics ; Retrospective Studies ; S100 Proteins ; metabolism ; Telomerase ; genetics