Expression Levels Of Cytokeratin Research Articles

ARID2 suppression promotes tumor progression and upregulates cytokeratin 8, 18 and β-4 integrin expression in TP53-mutated tobacco-related oral cancer and has prognostic implications.

Mutations in ARID2 and TP53 genes are found to be implicated in the tobacco related tumorigeneses. However, the effect of loss of ARID2 in the TP53 mutated background in tobacco related cancer including oral cancer has not been investigated yet. Hence, in this study we knockdown ARID2 using shRNA mediated knockdown strategy in TP53 mutated oral squamous cell carcinoma (OSCC) cell line and studied its tumorigenic role. Our study revealed that suppression of ARID2 in TP53 mutated oral cancer cells increases cell motility and invasion, induces drastic morphological changes and leads to a marked increase in the expression levels of cytokeratins, and integrins, CK8, CK18 and β4-Integrin, markers of cell migration/invasion in oral cancer. ARID2 suppression also showed early onset and increased tumorigenicity in-vivo. Interestingly, transcriptome profiling revealed differentially expressed genes associated with migration and invasion in oral cancer cells including AKR1C2, NCAM2, NOS1, ADAM23 and genes of S100A family in ARID2 knockdown TP53 mutated oral cancer cells. Pathway analysis of differentially regulated genes identified "cancer pathways" and "PI3K/AKT Pathway" to be significantly dysregulated upon suppression of ARID2 in TP53 mutated OSCC cells. Notably, decreased ARID2 expression and increased CK8, CK18 expression leads to poor prognosis in Head and Neck cancer (HNSC) patients as revealed by Pan-Cancer TCGA data analysis. To conclude, our study is the first to demonstrate tumor suppressor role of ARID2 in TP53 mutated background indicating their cooperative role in OSCC, and also highlights its prognostic implications suggesting ARID2 as an important therapeutic target in OSCC.

IMMUNOHISTOCHEMICAL AND MORPHOMETRIC CHARACTERISTICS OF CHORIONIC TROPHOBLAST AND DECIDUAL CELLS OF FETAL MEMBRANES IN CASE OF ANTE-INTRANATAL FETAL DEATH

The aim: To identify immunohistochemical and morphometric features of chorionic trophoblast cells and fetal membranes derived decidual cells, which were obtained from stillbirths associated with pre-eclampsia, iron deficiency anemia, and acute chorioamnionitis. Materials and methods: The study included 58 fetal membranes of fetuses, who died in the ante-intranatal period. The membranes were divided into 6 obstetric history-based groups: premature (n = 8) and full-term (n = 8) stillbirths complicated by preeclampsia; premature (n = 8) and full-term ( n = 8) stillbirths complicated by iron deficiency anemia, premature (n = 10) and full-term (n = 16) stillbirths complicated by chorioamnionitis. A control group consisted of 8 membranes obtained from physiological pregnancies followed by the birth of a live full-term baby. Samples (rupture site) were probed with cytokeratin to identify the fetal trophoblast layer of the chorion and with vimentin for further identification of the decidual cells. The thickness of the trophoblastic layer, expression levels of cytokeratin and vimentin were determined. Results: A decrease of the cytokeratin expression by the chorionic trophoblasts and a thinning of the chorionic trophoblast cell layer due to an increasing gestational age were shown in case of the mentioned pathological conditions. In comparison with the control group, the level of vimentin expression by decidual cells was increased in case of full-term pregnancy complicated by preeclampsia, decreased in pregnancies complicated by chorioamnionitis, and remained unchanged in case of accompanying anemia. Conclusions: The changes in the studied immunohistochemical parameters are more pronounced in case of chorioamnionitis, which indicates more severe morphological and functional changes.

Abstract 2524: MiR-29b may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells as well as on tumor cells

Abstract Background: Peritoneal metastasis (PM) frequently occur in patients with gastric cancer with poor prognosis. Peritoneal cavity is lined with monolayer of mesothelial cells (MC) and contains various types of cells and exosomes. MC play an important role in the development of PM through mesothelial-mesenchymal transition (MMT). MiR-29 family is well known as tumor suppressor. However, the role of miR-29s in PM is not well characterized. We have previously reported that miR-29 family in exosome derived from peritoneal fluid was downregulated in patients with peritoneal metastasis of gastric cancer. In this study, we investigated the functions of miR-29s in the process of peritoneal metastasis. Methods: Exosomes were purified from peritoneal fluids of 85 patients with gastric cancer and miR-29 levels in peritoneal exosomes were quantified with RT-PCR using TaqMan Advanced miRNA Assays. Proliferation and migration of human gastric cancer cell lines, MKN45 and NUGC, were examined with MTT and transwell assays, respectively. Human peritoneal mesothelial cells (HPMCs) were isolated by trypsinization of omental tissues obtained from patients undergoing bariatric surgery and cultured in DMEM supplemented with 20% fetal bovine serum. MiR-29b mimic and negative control were transfected by lipofection method using RNAiMAX. MMT was induced by the culture of HPMCs with 10 ng/ml TGF-β. Expressions of cytokeratin and vimentin were evaluated with flow cytometry. Results: The expressions of miR-29a, miR-29b and miR-29c in peritoneal exosomes were all downregulated in 33 patients with PM as compared to those without PM. Transfection of miR-29b mimic significantly decreased the proliferation of MKN45 and NUGC-4 by 22~58% as compared with negative control miRNA (p<0.01). Migration was inhibited more strongly by 84~90% (p<0.01). After 48 hour-culture with TGF-β, HPMCs apparently changed the morphology from round to spindle shape. The expression level of cytokeratin in HPMCs was decreased while vimentin tended to be upregulated by TGF-β. However, transfection of miR-29b, but not control miRNA, significantly suppressed the morphological changes and reduced the expression levels of vimentin with restored expression of cytokeratin, suggesting the inhibition of MMT in HPMCs. Conclusion: MiR-29b suppresses mesenchymal transition of MC as well as of tumor cells. Replacement of miR-29b to peritoneal cavity might be used for the treatment for peritoneal metastasis. Citation Format: Yuki Kimura, Hideyuki Ohzawa, Akira Saito, Naohiro Sata, Joji Kitayama. MiR-29b may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells as well as on tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2524.

Frequent expression of conventional endothelial markers in pleural mesothelioma: usefulness of claudin-5 as well as combined traditional markers to distinguish mesothelioma from angiosarcoma

ObjectivesDistinguishing pleural sarcomatoid mesotheliomas from true sarcomas is challenging because the former does not always express the mesothelial markers, and diagnosis is often made on the basis of keratin expression. Consequently, sarcomas such as angiosarcomas that express keratin complicate the differential diagnosis. Furthermore, some mesotheliomas have been reported to express endothelial markers. The aim of this study is to identify useful markers for distinguishing pleural sarcomatoid mesothelioma from angiosarcoma. Materials and methodsThis study enrolled 147 patients with pleural mesothelioma—93 with epithelioid, 25 with biphasic, and 29 with sarcomatoid subtypes—and 41 patients with angiosarcomas in various organs. The expression levels of cytokeratin, mesothelial, and endothelial markers were assayed in both groups to identify the markers that could assist in distinguishing mesothelioma from angiosarcoma. Cytokeratin (AE1/AE3, CAM 5.2), endothelial (CD31, CD34, ERG, factor VIII, and claudin-5), and mesothelial (calretinin, WT-1, podoplanin (D2-40), EMA, and CK5/6) markers were immunohistochemically assayed using tissue blocks. ResultsMore than 90% of the mesotheliomas and less than 20% of the angiosarcomas expressed cytokeratin. Calretinin was expressed in 82% of all types of mesotheliomas but in only 48% of sarcomatoid mesotheliomas. Endothelial markers were expressed in mesothelioma tissues—CD31 in 10.3%, CD34 in 3.5%, ERG in 29%, and factor VIII in 3.4%—and the positivity was higher in sarcomatoid than in epithelioid and biphasic mesotheliomas. Claudin-5 was expressed in all the angiosarcomas, but not in any of the mesotheliomas. ConclusionWe found overlapping immunophenotypes in pleural mesotheliomas and angiosarcomas, but the sensitivity and specificity of claudin-5 expression were sufficient to distinguish between them. The differential diagnosis of mesothelioma should therefore include claudin-5 in a panel of immunohistochemical markers to distinguish mesothelioma from angiosarcoma.

Tridax procumbens suppresses cellular markers of tumour growth, proliferation and invasion in pancreatic ductal adenocarcinoma cell line

Pancreatic ductal adenocarcinoma is one of the most fatal solid malignancies, with a 5‐year survival rate of approximately five percent. Hence, there is much interest in developing promising anticancer drugs. To address this issue, we cultured the pancreatic ductal adenocarcinoma cell line (Panc‐1) in supplemented Dulbecco’s Modified Eagle's medium at 37°C in a humidified incubator with 5% CO2. Panc‐1 cells were cultured without or with 20 μg/ml of the dichloromethane fraction (DCMF) of the ethanol leaf extract of Tridax procumbens. Dimethyl sulfoxide was used as control. We examined the expression levels of ductal (cytokeratin 7, peanut agglutinin), mesenchymal (vimentin), cell cycle and proliferation markers (Ki67, p53, p21Cip1/Wap‐1) by immunofluorescence. Confocal images were collected (n≥3) and processed with Image J and Zen 2.3 lite software. Data were analysed using student’s t‐test and results expressed as mean ±standard error of mean with P value <0.05 considered as significant.We observed a significant reduction (p<0.05) in the expression levels of cytokeratin 7, peanut agglutinin, vimentin and Ki67 proteins in DCMF‐treated Panc‐1 cells as compared to control cells. There was a significant increase (p<0.05) in the expression levels of p53 and p21Cip1/Wap‐1 proteins in DCMF‐treated Panc‐1 cells as compared to the control. In conclusion, overexpression of cytokeratin 7, peanut agglutinin, vimentin and Ki67, and reduced expression of p53 and p21Cip1/Wap‐1 in pancreatic cancer correlate with increased tumour growth, invasion and poor prognosis. Therefore, results from this study suggest that Tridax procumbens could be a potential therapeutic agent in the treatment of pancreatic ductal adenocarcinoma.Support or Funding InformationWe appreciate the support and funding received from the University of Ibadan, Ibadan, Nigeria, Commonwealth Scholarship Commission, United Kingdom and University of Bath, United Kingdom.

The overexpression of salivary cytokeratins as potential diagnostic biomarkers in head and neck squamous cell carcinomas.

BackgroundCytokeratin (CK) intermediate filaments are demonstrated to have enormous potential in regulating cellular motility and cancer progression. There are more than 20 divergent CKs that have been identified, of which CK 8, 17, 18 and 19 are reported to be elevated in the tumour biopsies of head and neck cancer squamous cell carcinoma (HNSCC) patients. However, CK expression profiles in the saliva of HNSCC patients have not been investigated. We aim to investigate the mRNA expression profiles of CKs in saliva collected from healthy controls, HPV-negative and -positive HNSCC patients.MethodsOral rinse samples were collected from 42 cancer-free healthy controls (age-matched) and patients who have been diagnosed with HPV-negative (n = 20) and -positive (n = 48) HNSCC.ResultsHere, we report that the mRNA expression profiles of CKs differed in saliva collected from healthy controls and HNSCC patients. The mRNA expression levels of CK 8 and 18 were significantly elevated in saliva collected from HPV-negative HNSCC patients; whilst, CK 17 and 19 were expressed at a higher mRNA level in saliva collected from HPV-positive HNSCC patients compared to healthy controls. Importantly, receiver operating characteristic (ROC) analysis showed salivary CK 8 and 18 to have superior sensitivity and specificity in discriminating the HPV-negative HNSCC patients from healthy controls (80% and 86%) as well as between HPV-negative and -positive HNSCC patients (75% and 81%).ConclusionIn summary, we have demonstrated that an aberrant expression of salivary CKs may serve as a potential non-invasive diagnostic biomarker in HNSCC.

Co-culture with endometrial stromal cells enhances the differentiation of human embryonic stem cells into endometrium-like cells.

In vitro differentiation of human embryonic stem cells (hESCs) into endometrium-like cells may provide a useful tool for clinical treatment. The aim of the present study was to investigate the differentiation potential of hESCs into endometrium-like cells using three methods, which included induction by feeder cells, co-culture with endometrial stromal cells and induction with embryoid bodies. Following differentiation, the majority of cells positively expressed cytokeratin and epithelial cell adhesion molecule (EPCAM). Factors associated with endometrium cell function, namely the estrogen and progesterone receptors (ER and PR), were also detected. At day 21 following the induction of differentiation, the expression levels of cytokeratin, EPCAM, ER and PR were significantly increased in the co-culture method group, as compared with the other two methods. Furthermore, these cells became decidualized in response to progesterone and prolactin. In addition, the number of cytokeratin-positive or EPCAM-positive cells significantly increased following the induction of differentiation using the co-culture method, as compared with the other two methods. The mRNA expression levels of Wnt members that are associated with endometrial development were subsequently examined, and Wnt5a was found to be significantly upregulated in the differentiated cells induced by feeder cells and co-culture with endometrial stromal cells; however, Wnt4 and Wnt7a expression levels were unaffected. Additionally, the mRNA expression levels of Wnt5a in the differentiated cells co-cultured with endometrial stromal cells were higher when compared with those induced by feeder cells. In conclusion, the present findings indicated that the co-culture system is the optimal protocol for the induction of hESC differentiation into endometrium-like cells, and Wnt5a signaling may be involved in this process.

O1-026 - Cytokeratin (CK) 8 Expression in Non-Small-Cell Lung Cancer (NSCLC) and its Relation to Tumor Invasiveness and Patient Survival

ABSTRACT Purpose Type II intermediate filament protein CK8 is highly expressed in NSCLC. The role of CKs in tumor invasiveness is controversial. While circulating CKs have been used as tumor markers, the significance of circulating CK8 in NSCLC is undetermined. The significance of CK8 in NSCLC was assessed using cell lines and clinical samples. Methods We quantitatively evaluated expression of type I CK 18/19/20, type II CK 7/8, type III vimentin, and type V lamins among 12 NSCLC, 7 SCLC and 10 non-LC lines. Highly invasive NSCLC sublines were selected from two adenocarcinoma cell lines, A549 and HI1017, using a membrane invasion chamber system (MICS), and were assessed for CK 7/8/18/19 expression. Exogenous CK19 was induced by transfection, and endogenous CK 8/18/19 were knocked down by RNA interference (RNAi), followed by invasion assay using MICS. Circulating CK8 was assessed using sera from 60 NSCLC and 10 SCLC patients and ELISA. Results and discussion CK 8/18 were expressed in all NSCLC lines and CK8 was the dominant type II CK. The invasive sublines of A549 and HI1017 showed lower expression levels of CKs compared with their parental cells. Suppression of either CK8 or CK18 by RNAi led to a decrease in the total CK amount and increased invasiveness of both HI1017 and A549 cells. On the other hand, serum levels of CK8 in NSCLC patients correlated with progressing tumor clinical stage, and inversely correlated with survival in patients with NSCLC. Apparently contradictory results in the roles of CK8 as a negative regulator of tumor invasion and its prognostic impact on poor survival in NSCLC may suggest that circulating CK8 may reflect tumor burden rather than invasiveness of individual tumors.

Ethyl acetate fraction of Radix rubiae inhibits cell growth and promotes terminal differentiation in cultured human keratinocytes

Ethnopharmacological relevanceIn Chinese medicine practice, Radix rubiae, the dry root of Rubia cordifolia L. is commonly used for the treatment of psoriasis. Aim of the studyPsoriasis is a chronic inflammatory skin disorder characterized by hyperproliferation and aberrant differentiation of epidermal keratinocytes. Our previous studies identified Radix rubiae to have potent antiproliferative action on cultured HaCaT keratinocytes and to induce keratinocyte differentiation in mouse tail model. The present study aimed to investigate whether Radix rubiae could also induce terminal differentiation in cultured human keratinocytes. Methods and resultsThe cornified envelope (CE) formation assay showed that ethyl acetate (EA) fraction of Radix rubiae significantly accentuated the CE formation, a well-recognized marker of terminal differentiation, in cultured HEK and HaCaT cells in a dose and time dependent manner. Western blot analyses demonstrated that EA fraction of Radix rubiae at a concentration of 3.2μg/ml significantly increased transglutaminase type I and involucrin expression in both HEK and HaCaT keratinocytes after 96h treatment, a response similar to that of Ca2+ positive control. Moreover, the expression level of cytokeratin 5/14, which is specifically related to cell proliferation, was significantly downregulated while terminal differentiation markers cytokeratin 1/10 were markedly increased by Radix rubiae treatment in both HEK and HaCaT cells. ConclusionThe present experimental findings unequivocally confirmed the keratinocyte terminal differentiation promoting capacity of Radix rubiae, and strongly suggest that Radix rubiae is a promising antipsoriatic agent warranting further clinical development for psoriasis treatment.

Cytometric comparisons between circulating tumor cells from prostate cancer patients and the prostate-tumor-derived LNCaP cell line

Many important experiments in cancer research are initiated with cell line data analysis due to the ease of accessibility and utilization. Recently, the ability to capture and characterize circulating tumor cells (CTCs) has become more prevalent in the research setting. This ability to detect, isolate and analyze CTCs allows us to directly compare specific protein expression levels found in patient CTCs to cell lines. In this study, we use immunocytochemistry to compare the protein expression levels of total cytokeratin (CK) and androgen receptor (AR) in CTCs and cell lines from patients with prostate cancer to determine what translational insights might be gained through the use of cell line data. A non-enrichment CTC detection assay enables us to compare cytometric features and relative expression levels of CK and AR by indirect immunofluorescence from prostate cancer patients against the prostate cancer cell line LNCaP. We measured physical characteristics of these two groups and observed significant differences in cell size, fluorescence intensity and nuclear to cytoplasmic ratio. We hope that these experiments will initiate a foundation to allow cell line data to be compared against characteristics of primary cells from patients.

Cytokeratins negatively regulate the invasive potential of lung cancer cell lines

Lung cancer cells express several cytokeratins (CKs) that are subdivided into type I (CK9-23) and type II (CK1-8) subclasses. The functions of CKs in lung cancer cells have not been fully elucidated. The purpose of this study was to investigate the role of CKs in the invasion of lung cancer cells. We investigated the expression levels of CK7, 8, 18 and 19 in 12 non-small cell lung cancer (NSCLC) and seven SCLC cell lines by quantitative immunoblotting. The expression levels of these four CKs were significantly higher in the NSCLC cells. The NSCLC cell line HI1017 expressed CK8 and 18; A549 cells expressed CK7, 8, 18 and 19, respectively. Invasive sublines of HI1017 and A549 were established by repeated selection of invasive cells using a membrane invasion chamber system. The invasive cell lines showed lower expression levels of CKs compared with the parental cells. Exogenous CK19 also resulted in a decrease in invasiveness of the HI1017 cells. Suppression of either CK8 or CK18 by short interfering RNAs led to a decrease in the total CKs and increased invasiveness of both the HI1017 and A549 cells. A549 cells expressed very low levels of CK19. Suppression of CK19 affected neither invasive ability nor total CK amount in the A549 cells. Our observations indicate that CK expression levels were inversely associated with invasiveness of the NSCLC cell lines, and suggest that expression levels of dominant CKs may affect invasive ability.

Dissemination profile of perioperative tumor cells in peripheral blood of colorectal cancer patients detected by multiple marker genes

This work proposes a method to assess the molecular profile of perioperative circulating tumor cells in peripheral blood (PB) of colorectal cancer patients for differentiating the dissemination process of tumor cells. Two-point quantification of multiple marker genes was designed for describing the profile. The expression levels of cytokeratin 20 (CK20), carcino-embryonic antigen (CEA) and survivin mRNA in PB and tumor tissue samples in 37 colorectal cancer patients from 1 d pre-operation to 2 h post-operation were detected with real-time quantitative reverse transcription-polymerase chain reaction. β-Actin mRNA was used as internal control to standardize the results of different mRNA expression levels. The data analysis using Stata statistical packages, Chi-Square test and Mann-Whitney test indicated the expression level of CEA mRNA in PB increased significantly, while those of CK20 and survivin mRNA decreased significantly. Quantitative comparison with tumor tissues indicated that the increase of CEA mRNA level in PB coincided with the decrease of CK20 and survivin mRNA levels in different tumor cells. These results showed surgical manipulation caused tumor cells shedding into blood from primary tumor tissue and significant increase of CEA mRNA level, while occult tumor cells with high expression levels of CK20 and survivin mRNA before surgery decreased after surgery.

Proteomic analysis to identify cytokeratin 18 as a novel biomarker of nasopharyngeal carcinoma

In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers. Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cytokeratin 18 in the above two tissues as well as 4 NPC cell lines was determined by Western blotting. Immunohistochemistry was also performed to detect the expression of cytokeratin 18 in 62 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. siRNA and in vitro cell invasion assay were used to check the correlation between the expression of cytokeratin 18 and invasive ability of NPC. Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cytokeratin 18 in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Significant cytokeratin 18 down-regulation was observed in NPC versus NNET (P = 0.000), whereas significant cytokeratin 18 up-regulation was observed in lymph node metastasis versus primary NPC (P = 0.001). In addition, cytokeratin 18 down-regulation was significantly correlated with poor histological differentiation (P = 0.000), whereas cytokeratin 18 up-regulation was significantly correlated with advanced clinical stage (P = 0.019), recurrence (P = 0.000), and regional lymph node metastasis (P = 0.001), and distant metastasis (P = 0.000). And down-regulated cytokeratin 18 expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Furthermore, survival curves showed that patients with cytokeratin 18 up-regulation had a poor prognosis (P = 0.000). Univariate analysis (Cox's proportional hazards model) showed that WHO histologic type (P = 0.025), lymph node metastasis (P = 0.007), distant metastasis (P = 0.005), recurrence (P = 0.000), and cytokeratin 18 (P = 0.000) were significantly associated with the prognosis of NPC. Multivariate analysis confirmed that lymph node metastasis (P = 0.012), distant metastasis (P = 0.009), recurrence (P = 0.006), and cytokeratin 18 (P = 0.001) were independent prognostic indicators. The data suggest that cytokeratin 18 is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC.

Detection of circulating tumor cells (CTCs) in patients with lung carcinoma by real-time fluorescent quantitative-PCR approach before and after chemotherapy

Circulating tumour cells (CTCs) are referred to the tumour cells that disseminated from the primary tumour and survive in circulating during the pro-ceeding of tumour growth. As surgical treatment evolves and local control has improved, the failure of cancer treatment has largely remained the re-sult of systemic metastasis. Selection of patients most likely to benefit from adjuvant strategies remains problematic. In order to develop a new standard of curative effect, this study was de-signed to track the number of CTCs in patients with lung cancer during chemotherapy. Methods: Samples of peripheral blood was taken from each lung cancer patients (n=32) on the day before chemotherapy as well as the third week after the chemotherapy cycle. The samples were subjected to real-time fluorescent quantitative reverse-tran- scriptase polymerase chain reaction (fqRT-PCR). Meanwhile the tumour size was determined by chest X-ray or computed tomograghy. Results Compared to that of pre-chemotherapy, the ex-pression level of cytokeratin (CK) 19 in the pa-tients significantly declined after chemotherapy (t=4.659,P=0.000). The level of CK19 mRNA in pa-tients with small cell lung cancer (SCLC) was higher than that of patients with non-small cell lung cancer (NSCLC) (t=1.944, P=0.061). The de-crease of CK19 mRNA level correlated well with the type during the treatment. Relatively the de-crease of SCLC is more obvious (t=6.073,P=0.000). The variation of CK19 mRNA level before and after chemotherapy was positively related to the dis-parity of tumour burden (r=0.593). There was also a significant association between the type (NSCLC vs. SCLC) and the change of tumour size (t=3.686, P=0.001).The positive rate before chemotherapy Supported by grant from the Natural Science Foundation in China (No.30972961). was 71.9% (23/32), while that after chemotherapy was 37.5% (12/32), indicating that 11 patients con- verted into negative after chemotherapy. Of the 16 patients which were in Ⅳ-stage, 11 cases were po- sitive (11/16,68.8%). Surprisingly, of the remaining 16 patients which were Ⅱ/Ⅲ stage, 12 cases were regarded as positive according to the criteria (12/6,75%). Conclusions: The real-time flu- ores-cent quantitative-PCR approach is useful for mea- suring the relative number of CTCs in a patients’ peripheral blood to monitor the effectiveness of treatment, and for designing more comprehensive and reasonable therapeutic regimes at earlier dates for patients. The treatment response can be immediately assessed by serial quantitation of CTCs after chemotherapy, and therefore this method highlights an alternative approach to rapidly access the patient’s response to treatment.

Estrogen receptor beta regulates epithelial cellular differentiation in the mouse ventral prostate.

We have previously reported epithelial cellular hyperplasia in ventral prostates (VP) of mice lacking estrogen receptor beta (ER beta). To investigate the causes of this phenomenon, we measured cellular proliferation and apoptosis in VP of ER beta(-/-) and WT mice. With BrdUrd labeling, the number of proliferating cells was 3.6-fold higher in ER beta(-/-) mice. There was also a decrease in apoptosis as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay and an increase in expression of the anti-apoptotic bcl-2. The state of differentiation of the epithelial cells of the VP was studied by immunohistochemical staining, Western blotting, and fluorescence-activated cell sorting (FACS). In ER beta(-/-) mouse VP, the number of p63-positive cells (basal phenotype) was 2.6-fold higher, and expression level of cytokeratin (CK) 8, a luminal cell marker, was lower. FACS analysis with p63 showed that in WT mice the ratio of basal to intermediate/luminal cell populations expressing p63 was 1:2.5, whereas in ER beta(-/-) mice it was 1:9. The expression of basal/intermediate marker CK 19 in three FACS areas, g1, g2, and g3, gated according to cellular size and granularity, was 1:0.6:2 in WT and 1:4:6.7 in ER beta(-/-) mice, showing a shift of CK 19-positive cells toward a cell population of intermediate size and granularity. We conclude that, in ER beta(-/-) mouse VP, there is increased epithelial proliferation, decreased apoptosis, and accumulation of incompletely differentiated cells in an intermediate pool. The continued proliferation of intermediate cells leads to the prostatic epithelial hyperplasia observed in the absence of ER beta signaling.

Molecular interaction between human tumor marker protein p150, the largest subunit of eIF3, and intermediate filament protein K7.

The human tumor marker protein p150 was identified as the largest subunit of eukaryotic translation initiation factor 3 (eIF3) (also known as p170/p180). Its expression level is not only upregulated in many transformed cell lines, but also in several human cancers including breast, cervical, esophageal, and stomach carcinomas. The function of p150 in cancer and initiation of translation are not well understood. Using the yeast two-hybrid genetic screen, we found that a portion of p150 interacts with hPrt1, another subunit of eIF3, and cytokeratin 7, an intermediate filament protein. The interactions between p150 and hPrt1, and between p150 and cytokeratin 7 were verified both in vivo and in vitro. The interaction site for hPrt1 was mapped to the carboxyl half of the coiled-coil region of the p150 protein between amino acids 664-835. The expression of hPrt1 was clearly upregulated in cancer tissue, similarly to that of p150. By contrast, no substantial difference in the expression level of cytokeratin 7 was observed between cancer and normal breast tissue, suggesting that cytokeratin 7 expression is not co-regulated with p150. Taken together, our studies suggest a new role for p150 in translation initiation, possibly by acting as an adapter molecule between the translation initiation apparatus and the cytoskeleton structure in the cell.

Intermediate filament protein expression in early developmental stages of the mouse. A confocal scanning laser microscopy study of in vitro fertilized and in vitro cultured pre-implantation mouse embryos.

Expression patterns of intermediate filament proteins have been studied during early mouse embryo development. For this purpose, pre-implantation embryos at different stages of development after in vitro fertilization were studied using antibodies to cytokeratins, vimentin and lamins, using the indirect immunofluorescence assay. The levels of expression were quantitated and localization of the protein constituents was assessed by means of confocal scanning laser microscopy. Our studies showed that, although the embryos grew in culture, vimentin could not be detected in a filamentous organization. Immunofluorescence for cytokeratins was only positive from the 8-cell stage onwards. In the morula stage an increased level of cytokeratin expression was observed with a transitional staining pattern, combining a filamentous and a diffuse occurrence. In the blastocyst stages profound cytokeratin filaments were seen in trophoblast cells but not in the inner cell mass. When the cytokeratin subtypes were analysed separately, it became apparent that expression levels of cytokeratins 8 and 18 increased gradually up to a filamentous pattern in the blastocyst stage. Cytokeratins 7 and 19, although elevated in the latter stage and showing a filamentous distribution, were not found as prominently as cytokeratins 8 and 18. A-type as well as B-type lamins could be detected in all developmental stages examined, as a faintly reactive nuclear lamina. In blastocysts both lamin types were detected in trophoblast as well as in inner cell mass.