CMTM6: A Critical Prognostic Indicator in Non-Small Cell Lung Cancer.

While CKLF-like MARVEL transmembrane domain containing 6 (CMTM6)'s role in stabilizing PD-L1 and immune evasion within tumors is established, its expression in lung cancer tissue and adjacent macrophages remains uncertain. The study aimed to elucidate this ambiguity by investigating CMTM6's role in non-small cell lung cancer (NSCLC) prognosis. Employing immunohistochemical staining on 141 NSCLC and 110 adjacent normal lung tissue samples, CMTM6 expression was evaluated using the HSCORE system. Interestingly, NSCLC exhibited significantly higher CMTM6 levels (161.04±86.60) compared to normal tissues (71.20±45.10) (p < 0.001), detected not only in cancer cells but also in macrophages, lymphocytes, and nearby bronchial epithelial cells. Stratifying patients by CMTM6 levels unveiled a correlation between heightened expression and poorer overall survival (p = 0.003), alongside a link to tumor-infiltrating lymphocytes (TIL) (p = 0.037), especially in cases with increased TIL. Multivariate analysis identified CMTM6 as an independent predictor of overall survival (p = 0.009), implying that elevated CMTM6 expression in NSCLC might signify an adverse prognostic marker for patient outcomes.

RGD-p21Ras-scFv expressed prokaryotically on a pilot scale inhibits ras-driven colorectal cancer growth by blocking p21Ras-GTP.

BACKGROUND: Ras gene mutation and/or overexpression are drivers in the progression of cancers, including colorectal cancer. Blocking the Ras signaling has become a significant strategy for cancer therapy. Previously, we constructed a recombinant scFv, RGD-p21Ras-scFv by linking RGD membrane-penetrating peptide gene with the anti-p21Ras scFv gene. Here, we expressed prokaryotically RGD-p21Ras-scFv on a pilot scale, then investigated the anti-tumor effect and the mechanism of blocking Ras signaling. METHODS: The E. coli bacteria which could highly express RGD-p21Ras-scFv was screened and grown in 100 L fermentation tank to produce RGD-p21Ras-scFv on optimized induced expression conditions. The scFv was purified from E. coli bacteria using His Ni-NTA column. ELISA was adopted to test the immunoreactivity of RGD-p21Ras-scFv against p21Ras proteins, and the IC50 of RGD-p21Ras-scFv was analyzed by CCK-8. Immunofluorescence colocalization and pull-down assays were used to determine the localization and binding between RGD-p21Ras-scFv and p21Ras. The interaction forces between RGD-p21Ras-scFv and p21Ras after binding were analyzed by molecular docking, and the stability after binding was determined by molecular dynamics simulations. p21Ras-GTP interaction was detected by Ras pull-down. Changes in the MEK-ERK /PI3K-AKT signaling paths downstream of Ras were detected by WB assays. The anti-tumor activity of RGD-p21Ras-scFv was investigated by nude mouse xenograft models. RESULTS: The technique of RGD-p21Ras-scFv expression on a pilot scale was established. The wet weight of the harvested bacteria was 31.064 g/L, and 31.6 mg RGD-p21Ras-scFv was obtained from 1 L of bacterial medium. The purity of the recombinant antibody was above 85%, we found that the prepared on a pilot scale RGD-p21Ras-scFv could penetrate the cell membrane of colon cancer cells and bind to p21Ras, then led to reduce of p21Ras-GTP (active p21Ras). The phosphorylation of downstream effectors MEK-ERK /PI3K-AKT was downregulated. In vivo antitumor activity assays showed that the RGD-p21Ras-scFv inhibited the proliferation of colorectal cancer cell lines. CONCLUSION: RGD-p21Ras-scFv prokaryotic expressed on pilot-scale could inhibited Ras-driven colorectal cancer growth by partially blocking p21Ras-GTP and might be able to be a hidden therapeutic antibody for treating RAS-driven tumors.

Cytokine-induced killer cells carrying recombinant oncolytic adenovirus expressing p21Ras scFv inhibited liver cancer.

Background: Oncolytic adenovirus-mediated gene therapy is an emerging strategy for cancer treatment. However, oncolytic adenoviruses are mainly administered locally at tumor site. Intravenous administration of oncolytic adenovirus for cancer gene therapy is a problem that needs to be solved urgently. Methods: We constructed recombinant oncolytic adenovirus KGHV500 carrying anti-p21Ras scFv and employed CIK cells to deliver KGHV500. TUNEL, wound healing, MTT, and Transwell invasion assays were used to determine the anti-tumor efficacy of KGHV500 on liver cancer cells in vitro. Nude mouse xenograft model was used to examine the anti-tumor efficacy of CIK cells combined with KGHV500 in vivo. Furthermore, KGHV500 accumulation in different organs was detected to assess the safety. Results: KGHV500 inhibited the migration, proliferation, invasion, and induced the apoptosis of liver cancer cells. CIK cells carrying KGHV500 reached tumor site and exerted much better anti-tumor efficacy than CIK cells or KGHV500 alone in nude mouse xenograft model. Moreover, we detected KGHV500 and anti-p21Ras scFv in different organs of nude mice, with little effects on the organs. Conclusions: We develop a novel strategy for the treatment of Ras-driven liver cancer by combining CIK cells with oncolytic adenovirus expressing anti-p21Ras scFv. Intravenous injection of CIK cells carrying KGHV500 in vivo significantly inhibits tumor growth, has little effect on normal organs, and is relatively safe.

RGD4C peptide mediates anti-p21Ras scFv entry into tumor cells and produces an inhibitory effect on the human colon cancer cell line SW480.

BACKGROUND: We prepared an anti-p21Ras scFv which could specifically bind with mutant and wild-type p21Ras. However, it cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate the scFv penetration into tumor cells and produce antitumor effects. METHODS: RGD4C-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed to express fusion proteins in E. coli, then the fusion proteins were purified with HisPur Ni-NTA. RGD4C-EGFP was used as reporter to test the factors affecting RGD4C penetration into tumor cell. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. The ability of RGD4C-p21Ras-scFv to penetrate SW480 cells and colocalization with Ras protein was detected by immunocytochemistry and immunofluorescence. The antitumor activity of the RGD4C-p21Ras-scFv was assessed with the MTT, TUNEL, colony formation and cell migration assays. Chloroquine (CQ) was used an endosomal escape enhancing agent to enhance endosomal escape of RGD4C-scFv. RESULTS: RGD4C-p21Ras-scFv fusion protein were successfully expressed and purified. We found that the RGD4C fusion protein could penetrate into tumor cells, but the tumor cell entry of was time and concentration dependent. Endocytosis inhibitors and a low temperature inhibited RGD4C fusion protein endocytosis into cells. The change of the cell membrane potential did not affect penetrability. RGD4C-p21Ras-scFv could penetrate SW480 cells, effectively inhibit the growth, proliferation and migration of SW480 cells and promote this cells apoptosis. In addition, chloroquine (CQ) could increase endosomal escape and improve antitumor activity of RGD4C-scFv in SW480 cells. CONCLUSION: The RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of ras-driven cancers.

Inhibition of glioma by adenovirus KGHV500 encoding anti-p21Ras scFv and carried by cytokine-induced killer cells.

Ras gene mutation or overexpression can lead to tumorigenesis in multiple kinds of cancer, including glioma. However, no drugs targeting Ras or its expression products have been approved for clinical application thus far. Adenoviral gene therapy is a promising method for the treatment of glioma. In this study, the human glioma cell line U251 was co-cultured with recombinant adenovirus KGHV500, and the anti-tumor effects of KGHV500 were determined by MTT, scratch test, Transwell invasion, and apoptosis assays. Then, KGHV500 was delivered via the intravenous injection of CIK cells into glioma xenografts. Tumor volume, ki67 proliferation index, apoptosis levels, and anti-p21Ras scFv expression were tested to evaluate targeting ability, anti-tumor efficacy, and safety. We found that the KGHV500 exhibited anti-tumor activity in U251 cells and increased the intracellular expression of anti-p21Ras scFv compared with that in the control groups. CIK cells delivered KGHV500 to U251 glioma cell xenografts and enhanced anti-tumor activity against glioma xenografts compared to that produced by the control treatment. In conclusion, targeting Ras is a useful therapeutic strategy for gliomas and other Ras-driven cancers, and the delivery of anti-p21Ras scFv by recombinant adenovirus and CIK cells may play an essential role in the therapy of Ras-driven cancers.

CD133 expressionand clinicopathologic significance in benign and malignant breast lesions.

BACKGROUND/OBJECTIVE: CD133 is the molecular marker of normal stem cells and progenitor cells and also confirmed as a marker for cancer stem cells in various tumors. The aim of this study is to examine the expression of CD133 and assess its clinicopathologic significance in benign and malignant breast lesions. METHODS: We analyzed the distribution of CD133 positive cells in breast usual ductal hyperplasia, atypical ductal hyperplasia (ADH), breast ductal carcinoma in situ (DCIS), and invasive breast carcinomas. We then explored the relationship between the CD133 expression and clinicopathologic features using immuno-histochemical staining. RESULTS: We found that CD133 is not expressed in the cells of normal breast tissue, but the expression rate increased with progression of lesions from usual hyperplasia, through atypical ductal hyperplasia, ductal carcinoma in situ and invasive carcinoma. The positive expression rate of CD133 in breast invasive ductal carcinoma correlated to histological grade, cancer stage, nodal status, metastasis, recurrence, event-free survival and overall survival. There was no significant correlation between CD133 expression and factors such as age, postmenopausal status, histological type, tumor size, estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 expression. CONCLUSION: CD133 may play an important role in the occurrence and development of breast cancer. CD133 positive breast cancer cells are closely related to invasiveness and its expression may predict a poor prognosis.

An interobserver reproducibility analysis of size-set semiautomatic counting for Ki67 assessment in breast cancer.

PURPOSE: The proliferation marker Ki67 has prognostic and predictive values in breast cancer, and the cutoff of the Ki67 label index (LI) is a key index for chemotherapy. However, poor interobserver consistency in Ki67 assessment has limited the clinical use of Ki67, especially in luminal cancers. Here, we reported a modified Ki67 assessment method, size-set semiautomatic counting (SSSAC) and investigated its interobserver reproducibility. METHODS: One hundred invasive breast cancer tissues were set immunostained for Ki67 in one laboratory, scanned as digital slides, and sent to 41 pathologists at the laboratories of 16 hospitals for Ki67 LI assessment using size-set semiautomatic counting (SSSAC), size-set visual assessment (SSVA) and size-set digital image analysis (SSDIA) with a specific image viewing software (Aperio Image Scope, Leica, Germany). The intraclass correlation coefficient (ICC) and Bland-Altman plot were used to evaluate interobserver reproducibility. The Wilcoxon signed-rank test was used to analyze the difference in the Ki67 values assessed by SSSAC and SSDIA. RESULTS: SSSAC demonstrated better interobserver reproducibility (ICC = 0.942) than SSVA (ICC = 0.802). The interobserver reproducibility was better in Ki67 homogeneously stained slides and centralized hot-spot slides than in scattered hot-spot slides. The Ki67 value assessed with SSSAC was obviously higher than that assessed with SSDIA (negative ranks (SSDIA < SSSAC): N = 80, sum of ranks = 4274.50; positive ranks (SSDIA > SSSAC): N = 17, sum of ranks = 478.50; Z = -6.837; P < 0.001). CONCLUSION: SSSAC shows satisfactory interobserver reproducibility in the Ki67 assessment of breast cancer and may be a candidate standard method for Ki67 LI assessment in breast cancer and other malignancies.

CIK cell-based delivery of recombinant adenovirus KGHV500 carrying the anti-p21Ras scFv gene enhances the anti-tumor effect and safety in lung cancer.

PURPOSE: Adenovirus (Ads) is one of the most popular vectors used in gene therapy for the treatment of cancer. However, systemic therapy is limited by circulating antiviral antibodies and poor viral delivery in vivo. In this study, we used cytokine-induced killer (CIK) cells as delivery vehicles of Ads KGHV500 carrying the anti-p21Ras scFv gene to treat Ras gene-related lung cancer and investigate the anti-tumor effect in vitro and in vivo. METHODS: The human lung cancer cell line A549 was employed to investigate the anti-tumor activity of recombinant Ads KGHV500 harboring the anti-p21Ras scFv gene using MTT, wound healing, transwell invasion, and apoptosis assays in vitro. Next, CIK cells were used as delivery vehicles to deliver KGHV500 carrying the anti-p21Ras scFv gene to treat A549-transplanted tumors in nude mice, and viral replication, p21Ras scFv expression, and the therapeutic efficacy were assessed. RESULTS: In vitro studies showed that KGHV500 had potent anti-tumor activity. In addition, in vivo, this combination therapy significantly inhibited the growth of lung cancer xenografts compared with mice treated with KGHV500 alone. KGHV500 and anti-p21Ras scFv were observed in tumor tissue, but were nearly undetectable in normal tissues. CONCLUSIONS: The co-delivery of anti-p21Ras scFv by CIK cells and KGHV500 could increase the anti-tumor effect and safety, and possess considerable advantages for the treatment of Ras-related cancer.

Anti-colorectal cancer effects of anti-p21Ras scFv delivered by the recombinant adenovirus KGHV500 and cytokine-induced killer cells.

BACKGROUND: Colorectal cancer (CRC) is the most common type of gastrointestinal cancer. CRC gene therapy mediated by adenovirus holds great promise for the treatment of malignancies. However, intravenous delivery of adenovirus exhibits limited anti-tumor activity in vivo when used alone. METHODS: In this study, the antitumor activity of the recombinant adenovirus KGHV500 was assessed with the MTT, TUNEL, Matrigel invasion and cell migration assays. To enhance the intravenous delivery of KGHV500 in vivo, cytokine-induced killer (CIK) cells were used as a second vector to carry KGHV500. We explored whether CIK cells could carry the recombinant adenovirus KGHV500 containing the anti-p21Ras single chain fragment variable antibody (scFv) gene into tumors and enhance antitumor potency. RESULTS: Our results showed that KGHV500 exhibited significant antitumor activity in vitro. In the nude mouse SW480 tumor xenograft model, the combination of CIK cells with KGHV500 could induce higher antitumor activity against colorectal cancer in vivo than that induced by either CIK or KGHV500 alone. After seven days of treatment, adenovirus and scFv were detected in tumor tissue but were not detected in normal tissues by immunohistochemistry. Therefore, KGHV500 replicates in tumors and successfully expresses anti-p21Ras scFv in a colorectal cancer xenograft model. CONCLUSIONS: Our study provides a novel strategy for the treatment of colorectal cancer by combining CIK cells with the recombinant adenovirus KGHV500 which carried anti-p21 Ras scFv.

Overexpression of wild-type p21Ras plays a prominent role in colorectal cancer.

Colorectal cancer (CRC) is the most common gastrointestinal type of cancer. The overexpression of Ras proteins, particularly p21Ras, are involved in the development of CRC. However, the subtypes of the p21Ras proteins that are overexpressed and the mutation status remain unknown restricting the development of therapeutic antibodies targeting p21Ras proteins. The present study aimed to investigate the mutation status of ras genes associated with Ras proteins that are overexpressed in CRC and explore whether or not wild-type p21Ras could be a target for CRC therapy. p21Ras expression was examined immunohistochemically in normal colorectal epithelium, benign lesions and malignant colorectal tumor tissues by monoclonal antibody (Mab) KGH-R1 which is able to react with three types of p21Ras proteins: H-p21Ras, N-p21Ras and K-p21Ras. Then, the expression levels of p21Ras subtypes were determined in CRC by a specific Mab for each p21Ras subtype. Mutation status of ras genes in p21Ras-overexpressing CRC was detected by DNA sequencing. There was rare p21Ras expression in normal colorectal epithelium but a high level of p21Ras expression in CRC, with a significant increase from normal colorectal epithelium to inflammatory polyps, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia and invasive colorectal adenocarcinoma, respectively. Overexpression of K-p21Ras was found in all CRC tissues tested, overexpression of N-p21Ras was found in 85.7% of the CRC tissues, while H-p21Ras expression was not found in any CRC tissue. DNA sequencing showed that there were no K-ras mutations in 60% of the K-p21Ras-overexpressing CRC, while 40% of the CRC tissues harbored K-ras mutations. N-ras mutations were not found in any N-p21Ras-overexpressing CRC. Our findings indicate that overexpression of wild-type p21Ras may play a prominent role in the development of CRC in addition to ras mutations and could be a promising target for CRC therapy.

A novel anti-p21Ras scFv antibody reacting specifically with human tumour cell lines and primary tumour tissues.

BACKGROUND: The ras genes play an important role in the development and progression of human tumours. Neutralizing Ras proteins in the cytoplasm could be an effective approach to blocking ras signalling. In this study, we prepared anti-p21Ras single chain fragment variable antibody (scFv) and investigated its immunoreactivity with human tumours. METHODS: The coding sequences of H-ras, K-ras, and N-ras were separately ligated into the vector pET-28a(+). Then, recombinant expressing plasmids were induced by IPTG for p21Ras expression in E. coli. Hybridoma cell lines producing anti-p21Ras monoclonal antibodies were isolated using wildtype p21Ras proteins as immunogens. Anti-p21Ras scFv antibody was prepared from the hybridoma by the phage scFv display method. The immunoreactivity of the anti-p21Ras monoclonal antibody and the scFv antibody was identified by ELISA and immunocytochemistry. RESULTS: We prokaryotically expressed wildtype H-p21Ras, K-p21Ras and N-p21Ras and generated the hybridoma cell line KGH-R1, producing anti-p21Ras monoclonal antibodies. It was demonstrated that KGH-R1 monoclonal antibody could recognize wildtype and mutated H-p21Ras, K-p21Ras and N-p21Ras in human tumour cell lines. In all 14 types of primary human cancer tissues tested, the monoclonal antibody presented strong immunoreactivity but showed weak or negative immunoreactivity in the corresponding normal tissues. Subsequently, we prepared anti-p21Ras scFv from hybridoma KGH-R1, which showed the same immunoreactivity as the original monoclonal antibody. Sequence analysis demonstrated that the nucleotides and amino acids of the scFv exhibited an approximately 50 % difference from the anti-p21Ras scFv reported previously. CONCLUSIONS: This study presents a novel anti-p21Ras scFv antibody. Our data suggest that the scFv may be useful for ras signalling blockage and may be a potential therapeutic antibody for ras-derived tumours.

The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo.

Activated ras genes are found in a large number of human tumors, and therefore are one of important targets for cancer therapy. This study investigated the antitumor effects of a novel single chain fragment variable antibody (scFv) against ras protein, p21Ras. The anti-p21Ras scFv gene was constructed by phage display library from hybridoma KGHR1, and then subcloned into replication-defective adenovirus vector to obtain recombinant adenovirus KGHV100. Human tumor cell lines with high expression of p21Ras SW480, MDA-MB­231, OVCAR-3, BEL-7402, as well as tumor cell line with low expression of p21Ras, SKOV3, were employed to investigate antitumor effects in vitro and in vivo. Fluorescence microscopy demonstrated that KGHV100 was able to express intracellularly anti-p21Ras scFv antibody in cultured tumor cells and in transplantation tumor cells. MTT, Transwell, colony formation, and flow cytometry analysis showed that KGHV100 led to significant growth arrest in tumor cells with high p21Ras expression, and induced G0/G1 cell cycle arrest in the studied tumor cell lines. In vivo, KGHV100 significantly inhibited tumor growth following intratumoral injection, and the survival rates of the mice were higher than the control group. These results indicate that the adenovirus-mediated intracellular expression of the novel anti-p21Ras scFv exerted strong antitumoral effects, and may be a potential method for therapy of cancers with p21Ras overexpression.

The antitumor effects of oncolytic adenovirus H101 against lung cancer.

Lung cancer is the leading cause of cancer mortality in both men and women, with dismal survival rates due to late-stage diagnoses and a lack of efficacious therapies. The new treatment options with completely novel mechanism of therapeutic activity are needed for lung cancer to improve patient outcome. The present study was aimed at testing the efficacy of recombinant adenovirus H101 as an oncolytic agent for killing human lung cancer cell lines in vitro and in vivo. We assessed the coxsackievirus adenovirus receptor (CAR) expression on human lung cancer cell lines by RT-PCR and immunocytochemistry staining. Viral infectivity and viral replication in lung cancer cells was assayed by flow cytometry and real-time fluorescent quantitative PCR. After H101 treatment, cytotoxic effect, cell cycle progression and apoptosis were further examined by lactate dehydrogenase release assay and flow cytometry in vitro, respectively. In vivo, antitumor effects of H101 were assessed on SCID Beige mice xenografted with human lung cancer cells. Receptor characterization confirmed that human lung cancer cell lines expressed CAR receptor for adenovirus type 5. Lung cancer cells were sensitive to infection by the H101 virus. H101 infection and replication resulted in very potent cytotoxicity, G2/M phase arrest and cell lysis. In vivo, we also showed that H101 significantly inhibited tumor growth following intratumoral injection, with virus replication, cell degeneration and necrosis in the tumor tissue. These results have important implications for the treatment of human lung cancer.

ER81-shRNA inhibits growth of triple-negative human breast cancer cell line MDA-MB-231 in vivo and in vitro.

The lack of effective treatment targets for triple-negative breast cancers make them unfitted for endocrine or HER2 targeted therapy, and their prognosis is poor. Transcription factor ER81, a downstream gene of the HER2, is highly expressed in breast cancer lines, breast atypical hyperplasia and primary breast cancers including triple-negative examples. However, whether and how ER81 affects breast cancer carcinogenesis have remained elusive. We here assessed influence on a triple-negative cell line. ER81-shRNA was employed to silence ER81 expression in the MDA-MB-231 cell line, and MTT, colony-forming assays, and flow cytometry were used to detect cell proliferation, colony-forming capability, cell cycle distribution, and cell apoptosis in vitro. MDA-MB-231 cells stably transfected with ER81-shRNA were inoculated into nude mice, and growth inhibition of the cells was observed in vivo. We found that ER81 mRNA and protein expression in MDA-MB-231 cells was noticeably reduced by ER81-shRNA, and that cell proliferation and clonality were decreased significantly. ER81-shRNA further increased cell apoptosis and the residence time in G0/G1 phase, while delaying tumor-formation and growth rate in nude mice. It is concluded that ER81 may play an important role in the progression of breast cancer and may be a potentially valuable target for therapy, especially for triple negative breast cancer.

[Immunoreactivity of monoclonal anti-p21Ras antibody KGH-R1 in colorectal benign and malignant lesions].

AIM: To investigate the immunoreactivity of monoclonal anti-p21ras antibody KGH-R1 in colorectal benign and malignant lesions. METHODS: Immunohistochemical staining was performed using monoclonal anti-p21ras antibody KGH-R1 prepared in our laboratory, in formalin-fixed, paraffin-embedded colorectal samples including normal colorectal tissues, colorectal inflammatory polyps, colorectal low-grade intraepithelial neoplasia, colorectal high-grade intraepithelial neoplasia, invasive colorectal carcinomas and corresponding adjacent tissues. Immunoreactivity of monoclonal antibody KGH-R1 was evaluated by percentage of positive cells and histological score (HSCORE). RESULTS: Immunostaining was found in 64.89% (61/94) of invasive colorectal adenocarcinomas with an average of 97.28% of carcinoma cells positive and average of 178.98 of HSCOREs. 60.24% (50/83) of colorectal high-grade intraepithelial neoplasia demonstrated immunostaining with KGH-R1, the average percentage of positive cells was 95.08%, the average HSCOREs was 156.38. 64.58% (31/48) of colorectal low-grade intraepithelial neoplasia demonstrated immunoreactivity with KGH-R1, the average percentage of positive cells was 82.52%, the average HSCOREs was 103.03. 39.97% (29/73) of colorectal inflammatory polyps showed immunoreactivity with KGH-R1, the average percentage of positive cells was 17.78%, the average HSCOREs was 18.66. 46.67% (21/45) of normal colorectal tissues showed immunostaining, but the immunoreactivity was very weak, the average percentage of positive cells was 2.64%, the average HSCOREs was 2.64. The the average percentage of positive cells and the average HSCOREs in invasive colorectal carcinomas had no statistical significance with adjacent high-grade intraepithelial neoplasia, but were higher than that in adjacent low-grade intraepithelial neoplasia. Weak immunostaining was found in 23.53% (20/85) of adjacent normal colorectal tissues. CONCLUSION: Suggested in this study that monoclonal anti-p21ras antibody KGH-R1 has a high immunoreactivity with invasive colorectal carcinomas and may be a potential therapeutic antibody in the future.

[The quantitative detection of NKG2D, perforin and granzyme B gene expressions in lymphocytes by SYBR green I real-time fluorescence quantitative PCR].

AIM: To develop a SYBR Green I real-time fluorescence quantitative PCR for the detection of lymphocyte immune function. METHODS: The primers of NKG2D, perforin, granzyme B and keeping-home gene GAPDH were designed and synthesized according to NCBI gene sequences, and the real-time fluorescence quantitative PCR was established. The gene expressions of NKG2D, perforin and granzyme B in lymphocytes from cancer patients and CIK induced from the cancer patients lymphocytes in vitro were quantified by real-time fluorescence quantitative PCR. RESULTS: NKG2D, perforin and granzyme B mRNA could be specifically amplified and quantitatively detected by the SYBR Green I real-time fluorescence quantitative PCR according to agarose gel electrophoresis and melt curve analysis. The mRNA expression of granzyme B was reduced in lymphocytes from cancer patients, however the mRNA expressions of perforin and granzyme B were increased in CIK induced by cytokines and monoclone antibody compared with their lymphocytes(P<0.01). CONCLUSION: The SYBR Green I real-time fluorescence quantitative PCR is a useful method for the quantitative detection of NKG2D, perforin and granzyme B mRNA to investigate cellular immune function.

[Promoter methylation and mRNA expression of WT1 gene in MCF10 breast cancer model].

OBJECTIVE: To investigate the role of WT1 gene in breast carcinogenesis by analyses of the promoter methylation status and mRNA expression of WT1 gene in MCF10 model system of breast cancer progression. METHODS: Methylation specific PCR and sodium bisufite genomic sequencing were employed to detect methylation status of WT1 promoter in normal breast tissue, traditional breast cancer cell line MCF7 and MCF10 model series, including MCF10A (breast hyperplastic cell line, non-tumorigenic), MCF10AT (pre-malignant cell line, forming slowly progressing hyper and dysplastic lesions), MCF10DCIS.com (breast ductal carcinoma in situ cell line, forming ductal carcinoma in situ), and three invasive cell lines with metastatic potential (MCF10CA1a, MCF10CA1d, and MCF10CA1h). Real time reverse transcription PCR assay was used to determine the mRNA expression levels of WT1 in various cell lines. RESULTS: Hypermethylation of WT1 promoter was identified in MCF7 and all MCF10 model cell lines (MCF10A, MCF10AT, MCF10DCIS.com, MCF10CA1a, MCF10CA1d, and MCF10CA1h). Unexpectedly, an increased expression of WT1 mRNA was found in all MCF10 cell lines and MCF7 comparing with normal breast tissue [folds of overexpression: 3.23 (MCF10A), 1.94 (MCF10AT), 4.20 (MCF10CA1a), 1.53 (MCF10CA1d), 4.20 (MCF10CA1h), 4.35 (MCF10DCIS) and 28.69 (MCF7)]. CONCLUSIONS: Promoter methylation does not silence the mRNA expression of WT1 during the development of breast cancer. Overexpression of WT1 occurs in the early stages of breast cancer development, suggesting its role as an oncogene rather than a tumor suppressor gene.

[Promoter methylation and mRNA expression of APC gene in MCF10 breast cancer model].

OBJECTIVE: To investigate the promoter methylation status and mRNA expression of APC gene in MCF10 model of breast cancer progression. METHODS: Methylation specific PCR and sodium bisufite genomic sequencing were employed to detect the methylation status of APC promoter 1A in normal breast tissues, conventional breast cancer cell line MCF-7 and MCF10 model cell lines including MCF10A (breast hyperplastic cell line, non-tumorigenic), MCF10AT (pre-malignant cell lines, producing slowly progressing hyperplastic and dysplastic lesions), MCF10DCIS.com (breast ductal carcinoma in-situ cell line, producing ductal carcinoma in-situ), MCF10CA1a, MCF10CA1d, MCF10CA1h cell lines (invasive breast carcinoma cell line, forming aggressive tumors of different morphology and metastatic potential). In addition, mRNA expression of APC was determined by reverse transcriptase PCR and real-time PCR assays. RESULTS: Hypomethylation of APC promoter 1A was identified in hyperplastic cell line MCF10A, pre-malignant cell line MCF10AT, ductal carcinoma in-situ cell line MCF10DCIS.com, invasive carcinoma cell lines MCF10CA1a, MCF10CA1d, MCF10CA1h and normal breast tissue. MCF-7 showed partial methylation at the promoter. Statistically significant reduction of APC mRNA expression was not found in all MCF10 cell lines and MCF-7, compared with that of normal breast tissue (MCF10AT, MCF10CA1a, MCF10CA1d, MCF10CA1h and MCF10DCIS.com showed reduced mRNA expressions of APC at 0.27, 0.96, 1.78, 2.70, and 2.03 times respectively. MCF10A and MCF-7 even showed an increase of APC mRNA expression at 0.02 and 0.33 times, respectively). CONCLUSION: The aberrant promoter methylation of APC is not related to the breast cancer progression, at least in the MCF10 model system.