PUMC-MB1 is a novel group 3 medulloblastoma preclinical model, sensitive to PI3K/mTOR dual inhibitor.

PURPOSE: Medulloblastoma (MB), a common and heterogeneous posterior fossa tumor in pediatric patients, presents diverse prognostic outcomes. To advance our understanding of MB's intricate biology, the development of novel patient tumor-derived culture MB models with necessary data is still an essential requirement. METHODS: We continuously passaged PUMC-MB1 in vitro in order to establish a continuous cell line. We examined the in vitro growth using Cell Counting Kit-8 (CCK-8) and in vivo growth with subcutaneous and intracranial xenograft models. The xenografts were investigated histopathologically with Hematoxylin and Eosin (HE) staining and immunohistochemistry (IHC). Concurrently, we explored its molecular features using Whole Genome Sequencing (WGS), targeted sequencing, and RNA sequecing. Guided by bioinformatics analysis, we validated PUMC-MB1's drug sensitivity in vitro and in vivo. RESULTS: PUMC-MB1, derived from a high-risk MB patient, displayed a population doubling time (PDT) of 48.18 h and achieved 100% tumor growth in SCID mice within 20 days. HE and Immunohistochemical examination of the original tumor and xenografts confirmed the classification of PUMC-MB1 as a classic MB. Genomic analysis via WGS revealed concurrent MYC and OTX2 amplifications. The RNA-seq data classified it within the Group 3 MB subgroup, while according to the WHO classification, it fell under the Non-WNT/Non-SHH MB. Comparative analysis with D283 and D341med identified 4065 differentially expressed genes, with notable enrichment in the PI3K-AKT pathway. Cisplatin, 4-hydroperoxy cyclophosphamide/cyclophosphamide, vincristine, and dactolisib (a selective PI3K/mTOR dual inhibitor) significantly inhibited PUMC-MB1 proliferation in vitro and in vivo. CONCLUSIONS: PUMC-MB1, a novel Group 3 (Non-WNT/Non-SHH) MB cell line, is comprehensively characterized for its growth, pathology, and molecular characteristics. Notably, dactolisib demonstrated potent anti-proliferative effects with minimal toxicity, promising a potential therapeutic avenue. PUMC-MB1 could serve as a valuable tool for unraveling MB mechanisms and innovative treatment strategies.

High-throughput functional screen identifies YWHAZ as a key regulator of pancreatic cancer metastasis.

Pancreatic cancer is a leading cause of cancer death due to its early metastasis and limited response to the current therapies. Metastasis is a complicated multistep process, which is determined by complex genetic alterations. Despite the identification of many metastasis-related genes, distinguishing the drivers from numerous passengers and establishing the causality in cancer pathophysiology remains challenging. Here, we established a high-throughput and piggyBac transposon-based genetic screening platform, which enables either reduced or increased expression of chromosomal genes near the incorporation site of the gene search vector cassette that contains a doxycycline-regulated promoter. Using this strategy, we identified YWHAZ as a key regulator of pancreatic cancer metastasis. We demonstrated that functional activation of Ywhaz by the gene search vector led to enhanced metastatic capability in mouse pancreatic cancer cells. The metastasis-promoting role of YWHAZ was further validated in human pancreatic cancer cells. Overexpression of YWHAZ resulted in more aggressive metastatic phenotypes in vitro and a shorter survival rate in vivo by modulating epithelial-to-mesenchymal transition. Hence, our study established a high-throughput screening method to investigate the functional relevance of novel genes and validated YWHAZ as a key regulator of pancreatic cancer metastasis.

Establishment and characterization of a new human colon cancer cell line, PUMC-CRC1.

Colorectal cancer (CRC) is one of the most common and fatal gastrointestinal cancers worldwide. Considering their diversity, the establishment of new continuous CRC cell lines with clear genetic backgrounds will provide useful tools for exploring molecular mechanisms, screening and evaluating antitumor drugs in CRC studies. Our de novo CRC cell line, PUMC-CRC1 (Peking Union Medical College Colorectal Cancer 1) was derived from a 47-year-old Chinese female patient diagnosed with moderately to poorly differentiated colon adenocarcinoma. Multiple experiments were used for full characterization. The new cell line was epithelial-like and was passaged for more than 40 times, with a population doubling time of 44 h in vitro, detected by cell counts. The cells exhibited complicated chromosomal abnormalities. The tumor formation rate in SCID mice was 100%. The xenograft tumor was adenocarcinoma with poor to moderate differentiation by Haematoxylin and Eosin staining (H&E) sections. Immunohistochemistry (IHC) analysis and next-generation sequencing (NGS) revealed microsatellite stable (MSS), APC (p.T1493fs) inactivation, KRAS (p.G12V) activation, and SMAD4 (p.V506A) mutation. Quality control of the cell line proved mycoplasma negative and identical STR profile with that of the original tissue, and no interspecific or intraspecific cross contamination was detected. In conclusion, PUMC-CRC1 was a newly established and well characterized human colon cancer cell line, which might be a good model for both in vitro and in vivo studies of the mechanism of colon cancer progression and the treatment strategies for MSS CRC.

The cell line ontology-based representation, integration and analysis of cell lines used in China.

BACKGROUND: The Chinese National Infrastructure of Cell Line stores and distributes cell lines for biomedical research in China. This study aims to represent and integrate the information of NICR cell lines into the community-based Cell Line Ontology (CLO). RESULTS: We have aligned, represented, and added all identified 2704 cell line cells in NICR to CLO. We also proposed new ontology design patterns to represent the usage of cell line cells as disease models by inducing tumor formation in model organisms, and the relations between cell line cells and their expressed or overexpressed genes or proteins. The resulting CLO-NICR ontology also includes the Chinese representation of the NICR cell line information. CLO-NICR was merged into the general CLO. To serve the cell research community in China, the Chinese version of CLO-NICR was also generated and deposited in the OntoChina ontology repository. The usage of CLO-NICR was demonstrated by DL query and knowledge extraction. CONCLUSIONS: In summary, all identified cell lines from NICR are represented by the semantics framework of CLO and incorporated into CLO as a most recent update. We also generated a CLO-NICR and its Chinese view (CLO-NICR-Cv). The development of CLO-NICR and CLO-NIC-Cv allows the integration of the cell lines from NICR into the community-based CLO ontology and provides an integrative platform to support different applications of CLO in China.

PKCßII-induced upregulation of PGP9.5 and VEGF in postoperative persistent pain in rats.

PURPOSE: Postoperative pain is a common clinical problem. In this study, we aimed to investigate the role of protein kinase C ßII (PKCßII) in the progression of postoperative pain following skin/muscle incision and retraction (SMIR) surgery. MATERIALS AND METHODS: SMIR postoperative pain model was established in rats, akin to a clinical procedure. The expression level and location of p-PKCßII were observed in dorsal root ganglion (DRG) or spinal cord from SMIR-operated rats by Western blotting and immunofluorescence. In addition, the effects of PKCßII on the expression of protein gene product 9.5 (PGP9.5) or vascular endothelial growth factor (VEGF) were assessed by using pharmacological activator and inhibitor of PKCßII. Moreover, mechanical withdrawal threshold (MWT) was assessed before or after SMIR-operated rats were treated with inhibitor or activator of PKCßII. RESULTS: The expression of PKCßII in DRG and spinal cord was significantly increased after SMIR surgery (P < 0.001, P < 0.01) and expression of PKCßII was located in the neurons of the spinal cord, and magnocellular neurons, non-peptide neurons, and peptide neurons in DRG. Besides, compared with skin/muscle incision group, retraction caused a marked increase in the expression of PKCßII and a significant decrease of MWT (P < 0.001, P < 0.05). The activator of PKCßII greatly increased the expression of PGP9.5 and VEGF (P < 0.05, P < 0.01) and enhanced MWT (P < 0.001), while inhibitor of PKCßII decreased the expression of PGP9.5 and VEGF and attenuated MWT (P < 0.05, P < 0.01, P < 0.001). CONCLUSION: Activation of PKCßII signaling pathways might be an important mechanism in the progression of postoperative pain.

ALDH1A3 affects colon cancer in vitro proliferation and invasion depending on CXCR4 status.

BACKGROUND: Aldehyde dehydrogenase (ALDH) has been widely used as a marker of cancer stem cells (CSCs). However, the ALDH family includes 19 members, and the most relevant isoforms and their biological functions in cancer biology are still controversial. METHODS: We examined ALDH enzyme activity and the mRNA expression of 19 ALDH members in 58 human cell lines. The biological effect and mechanism of knocking down ALDH1A3 with siRNA and shRNA in cell lines were explored. Finally, the relationship between ALDH1A3 and CXCR4 was analysed in a large panel of cell lines. RESULTS: ALDH1A3 is the key isoform that contributed to Aldefluor positivity in cell lines. Knocking down ALDH1A3 in different cancer cells conferred opposite phenotypes due to differential effects on CXCR4 expression. There was a significant negative correlation between ALDH1A3 and CXCR4 in 58 human cell lines. CONCLUSIONS: ALDH1A3 was the main contributor to Aldefluor positivity in human cell lines, and its contrasting effects might arise from differences in CXCR4 expression.

A Combination of Species Identification and STR Profiling Identifies Cross-contaminated Cells from 482 Human Tumor Cell Lines.

Human tumor cell lines are extremely important tools for cancer research, but a significant percentage is cross-contaminated with other cells. Short tandem repeat (STR) profiling is the prevailing standard for authenticating cell lines that originate from human tissues. Based on the analysis of 482 different human tumor cell lines used in China by STR, up to 96 cell lines were misidentified. More importantly, the study has found that STR profiling alone is insufficient to exclude inter-species cross-contamination of human cell lines. Among the 386 cell lines which had a correct STR profile, 3 of them were inter-species cross-contaminated. Careful microscopic examination may be helpful in some cases to detect changes in morphology but additional testing is needed. Additionally, species verification by PCR could easily identify the contaminants, even with a low percentage of contaminating cells. Combining STR profiling with species identification by PCR, more than 20.5% (99/482) of tumor cell lines were revealed as having been incorrectly identified, including intra-species (14.5%), inter-species (4.4%) cross-contamination and contaminating cell lines (1.7%). Therefore, quality control of cell lines is a systemic issue. Each cell line should undergo a full QA (Quality Assurance) assessment before it is used for research.

Divergent Effect of Dezocine, Morphine and Sufentanil on Intestinal Motor Function in Rats.

BACKGROUND: Opioid induced bowel dysfunction is the most common side effect of preoperatively administrated morphine, fentanyl and its derivative. However, the influence of dezocine on intestinal mobility is rarely reported. This study was designed to investigate the effects of dezocine, morphine and sufentanil on both intestinal smooth muscle contraction and propulsion in rats. METHODS: Contractile tension and frequency of isolated rat small intestine smooth muscle were measured using tension transducer after incubation with different concentrations of dezocine, morphine and sufentanil. The propulsive rate of methylene blue in rat intestinal tract was measured 30 minutes after intraperitoneal injection of morphine, sufentanil and dezocine. Percent of change in contractile tension and contraction frequency compared to baseline level were calculated to evaluate muscle contraction. Propulsive rate of methylene blue was calculated as the percentage of methylene blue moving distance in intestinal tract compared to the length of the small intestine. RESULTS: Morphine and sufentanil significantly increased the contractile tension of isolated small intestine smooth muscle at high doses. The contraction frequency did not change significantly among the 3 tested doses. Increasing the dose of dezocine from 1.7 mg.L(-1) to 10.2 mg.L(-1) did not change either the contractile tension or the contraction frequency. The propulsive rate of methylene blue in intestinal tract was significantly decreased after the treatment with morphine, sufentanil and dezocine (45.6%, 43.7%, and 42.1% respectively) compared to control group(57.1%), while the difference among the 3 drug groups were not significant. CONCLUSION: Morphine and sufentanil may dose dependently increase the contractile tension and contraction ability of isolated rat small intestine smooth muscle, while dezocine has no significant effect on intestine smooth muscle contraction. However, all these opioids might impair small intestinal propulsion.

Cloning and expression of a novel target fusion protein and its application in anti-tumor therapy.

BACKGROUNDS: Epidermal growth factor (EGF) is a 53 amino acid polypeptide and its receptor EGFR is an established therapeutic target for anti-tumor therapy. Two major categories of EGFR-targeted drugs include monoclonal antibodies (mAbs) and small molecular tyrosine kinase inhibitors (TKIs). However, drug resistance occurs in a significant proportion of patients due to EGFR mutations. Since EGFR can maintain activation while abrogating the activity of mAbs or TKIs, or bypass signaling functions while successfully circumventing the EGF-EGFR switch, developing new mechanism-based inhibitors is necessary. METHODS: In this study, based on the principle of tumor immunotherapy, a recombinant protein pLLO-hEGF was constructed. The N-terminal portion contains three immunodominant epitopes from listeriolysin O (LLO) and the C-terminal includes EGF. To use EGF as a target vector to recognize EGFR-expressing cancer cells, immunodominant epitopes could enhance immunogenicity of tumor cells for immune cell activation and attack. RESULTS: Recombinant protein pLLO-hEGF was successfully expressed and showed strong affinity to cancer cells. Also, pLLO-hEGF could significantly stimulate human lymphocyte proliferation and the lymphocytes demonstrated enhanced killing potency in EGFR-expressing cancer cells in vitro and in vivo. CONCLUSION: This study can provide novel strategies and directions in tumor biotherapy.

Ubiquitin-specific peptidase 22 inhibits colon cancer cell invasion by suppressing the signal transducer and activator of transcription 3/matrix metalloproteinase 9 pathway.

Colon cancer is associated with increased cell migration and invasion. In the present study, the role of ubiquitin-specific peptidase 22 (USP22) in signal transducer and activator of transcription 3 (STAT3)-mediated colon cancer cell invasion was investigated. The messenger RNA levels of STAT3 target genes were measured by reverse transcription-quantitative polymerase chain reaction, following USP22 knockdown by RNA interference in SW480 colon cancer cells. The matrix metalloproteinase 9 (MMP9) proteolytic activity and invasion potential of SW480 cells were measured by zymography and Transwell assay, respectively, following combined USP22 and STAT3 short interfering (si)RNA treatment or STAT3 siRNA treatment alone. Similarly, a cell counting kit-8 assay was used to detect the proliferation potential of SW480 cells. The protein expression levels of USP22, STAT3 and MMP9 were detected by immunohistochemistry in colon cancer tissue microarrays (TMAs) and the correlation between USP22, STAT3 and MMP9 was analyzed. USP22/STAT3 co-depletion partly rescued the MMP9 proteolytic activity and invasion of SW480 cells, compared with that of STAT3 depletion alone. However, the proliferation of USP22/STAT3si-SW480 cells was decreased compared with that of STAT3si-SW480 cells. USP22 expression was positively correlated with STAT3 and MMP9 expression in colon cancer TMAs. In conclusion, USP22 attenuated the invasion capacity of colon cancer cells by inhibiting the STAT3/MMP9 signaling pathway.

Ubiquitin-specific peptidase USP22 negatively regulates the STAT signaling pathway by deubiquitinating SIRT1.

BACKGROUND/AIMS: The ubiquitin-specific peptidase USP22 mediates various cellular and organismal processes, such as cell growth, apoptosis, and tumor malignancy. However, the molecular mechanisms that regulate USP22 activity remain poorly understood. Here we identify STAT3 as a new USP22 interactor. METHODS: · We used western blotting and RT-PCR to measure key protein, acetylated STAT3, and mRNA levels in HEK293 and colorectal cancer cell lines transfected with expression plasmids or specific siRNAs. Co-immunoprecipitation was used to demonstrate protein-protein interaction and protein complex composition. RESULTS: USP22 overexpression down-regulated STAT3 acetylation by deubiquitinating SIRT1. The three proteins were found to be present in a single protein complex. SiRNA-mediated depletion of endogenous USP22 resulted in SIRT1 destabilization and elevated STAT3 acetylation. Consistent with this finding, USP22 also down-regulated the expression of two known STAT3 target genes, MMP9 and TWIST. CONCLUSION: We show that USP22 is a new regulator of the SIRT1-STAT3 signaling pathway and report a new mechanistic explanation for cross talk between USP22 and the SIRT1-STAT pathways.

[Enhanced growth inhibition by combined two pathway inhibitors on K-ras mutated non-small cell lung cancer cells].

OBJECTIVE: To evaluate the effect of combined targeting of MEK and PI3K signaling pathways on K-ras mutated non-small cell lung cancer cell line A549 cells and the relevant mechanisms. METHODS: A549 cells were treated with different concentrations of two inhibitors. Growth inhibition was determined by MTT assay. According to the results of MTT test, the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941,0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244+0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244+5.0 µmol/L GDC-0941). The cell cycle and apoptosis were analyzed by flow cytometry. The expression of proteins related to apoptosis was tested with Western blot. RESULTS: Both GDC-0941 and AZD6244 inhibited the cell proliferation. The combination group II led to a stronger growth inhibition. The combination group I showed an antagonistic effect and combination group II showed an additive or synergistic effect. Compared with the control group, the combination group I led to reduced apoptotic rate [(20.70 ± 0.99)% vs. (18.65 ± 0.92 )%, P > 0.05]; Combination group II exhibited enhanced apoptotic rate [(37.85 ± 3.18)% vs. (52.27 ± 4.36)%, P < 0.01]. In addition, in the combination group II, more A549 cells were arrested in G0/G1 phase and decreased S phase (P < 0.01), due to the reduced expressions of CyclinD1 and Cyclin B1, the increased cleaved PARP and the diminished ratio of Bcl-2/Bax. CONCLUSIONS: For single K-ras mutated NSCLC cell line A549 cells, combination of RAS/MEK/ERK and PI3K/AKT/mTOR inhibition showed synergistic effects depending on the drug doses. Double pathways targeted therapy may be beneficial for these patients.

[Growth inhibition of combined pathway inhibitors on KRAS mutated non-small cell lung cancer cell line].

OBJECTIVE: To investigate the effect of the selective PI3K inhibitor and MEK inhibitor on KRAS and PTEN co-mutated non-small cell lung cancer cell line NCI-H157 and the relevant mechanisms. METHODS: NCI-H157 was cultured routinely and treated with different concentrations of the two inhibitors. Cell proliferation was detected by MTT cell cycle assay. Based on the MTT results the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941, 0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244 + 0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244 + 5.0 µmol/L GDC-0941). Colony formation assay was performed to detect colony formation efficiency. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of protein related to apoptosis was tested with Western blot. RESULTS: Cell growth was inhibited by the two inhibitors. Combination groups led to stronger cell proliferation inhibition: combination group Ishowed synergistic effect of their actions and combination group II showed an additive effect; in both groups, there were decreased colony number [(77.2 ± 1.54)/well vs (61.50 ± 2.12)/well, P < 0.01] and [(51.00 ± 4.00)/ well vs (22.50 ± 3.53)/well, P < 0.01]; and enhanced apoptotic ratios [(18.30 ± 0.82)% vs (21.32 ± 0.56)%, P < 0.01] and [(27.14 ± 1.58)% vs (42.45 ± 4.42)%, P < 0.01]. In addition, compared to the PI3K inhibitor alone group, the NCI-H157 cells in the combination groups showed increased G0/G1 phase and decreased S phase (P < 0.01). Western blotting showed that the combination groups demonstrated significantly decreased expression of cyclin D1 and cyclin B1, increased p21 and cleaved PARP and decreased bcl-2/bax ratio, compared to the PI3K inhibitor only group. CONCLUSION: The combined inhibition of PI3K (AZD6244) and MEK (GDC-0941) has synergistic effects on the proliferation of NCI-H157 cells, but such effects appear to be in a dose-dependent manner.

Generation and characterization of virus-free reprogrammed melanoma cells by the piggyBac transposon.

PURPOSE: Reprogramming of cancer cells to stem cell-like state provides a promising tool for the study of cancer pathogenesis and drug screening. However, most instances of direct reprogramming have been achieved by forced co-expression of defined transcription factors using viral vectors. Retroviral transduction as well as the ectopic expression of reprogramming factors may alter the differentiation potential of reprogrammed cancer cells or induce malignant transformation. Therefore, generation of reprogrammed cancer cells via virus-free reprogramming strategy needs to be studied. METHODS: Melanoma cells were reprogrammed by co-expression of doxycycline-inducible Oct4, Sox2, Klf4, and c-Myc using the piggyBac (PB) transposon system. The expression level of genes was analyzed through RT-PCR, Western blot, and immunofluorescence. Epigenetic modification of genes was detected by bisulfite genomic sequencing. Post reprogrammed melanoma cells were generated through differentiation of reprogrammed melanoma cells. Sensitivity to chemotherapeutic agents and metastasis potential were investigated in post reprogrammed melanoma cells. RESULTS: The virus-free reprogrammed melanoma cells were positive for stem cell markers including Oct4, Nanog, and SSEA-1, and the promoters of Nanog and Oct4 were demethylated. Moreover, reprogrammed melanoma cells gained differentiation potential and higher sensitivity to differentiation-inducing drugs. Post reprogrammed melanoma cells showed lower proliferation rate and metastatic potential compared with the parental cells. CONCLUSIONS: Our results indicate that PB transposon-based method is applicable to generate virus-free reprogrammed melanoma cells. These cells can differentiate into other lineages with loss of malignant phenotypes, which may provide a more suitable source for molding of cancer pathogenesis.

CD133 affects the invasive ability of HCT116 cells by regulating TIMP-2.

CD133 is widely expressed in colorectal cancer (CRC) tissues and cell lines. This protein has been used as a marker of CRC cancer stem cells, although the function and mechanism of CD133 in CRC invasion and metastasis remain unclear. In our study, we examined the role of CD133 in CRC invasion in vitro and investigated the mechanism involved in CD133-related invasion. CD133(high) and CD133(low) HCT116 cells were isolated, and the proliferation and invasive ability of these two subpopulations were tested. CD133(high) HCT116 cells exhibited greater proliferation and invasion compared with CD133(low) HCT116 cells. CD133 knockdown (using CD133 small-interfering [si]RNA) inhibited the invasive activity of CD133si-HCT116 cells. For the first time, we found that the expression of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) was down-regulated in CD133si-HCT116 cells. In addition, for the TIMP-2si-HCT116 cells (transfected with TIMP-2 siRNA), in vitro invasion was significantly decreased, whereas the expression of CD133 remained unchanged. Finally, the metalloproteinase 2 and MEK/ERK signaling pathways were examined, and no significant change was observed after the knockdown of CD133 or TIMP-2 in HCT116 cells. In conclusion, we demonstrated that CD133 plays an important role in HCT116 cell invasion, and for the first time, we found that CD133 knockdown significantly down-regulated TIMP-2 expression, which suggests that CD133 affects the invasive ability of HCT116 cells by regulating TIMP-2.

Fusion between tumor cells enhances melanoma metastatic potential.

PURPOSE: Malignant melanoma, characterized by early distant metastasis to the lungs and brain, is a leading cause of mortality related to skin cancer. Cell fusion and the subsequent aneuploidy, commonly observed in melanoma, are associated with poor prognosis. However, the pathological consequences of cell fusion in melanoma remain unknown. Therefore, the present study aims to investigate the pathological consequences of cell fusion in melanoma and the mechanism of melanoma metastasis. METHODS: Phytohemagglutinin-polyethylene glycol (PHA-PEG) fusion method was developed for the fusion of tumor cells. Melanoma cells were fused through the improved PHA-PEG fusion method and obtained by fluorescence-activated cell sorting. DNA content was analyzed through flow cytometry. Cell proliferation rate was detected by cell culture in vitro, and the cell number was counted daily. To detect the tumor growth rate in vivo, cells were injected subcutaneously and the tumor volumes were measured using a vernier caliper. To analyze the tumor metastatic potential, cells were injected intravenously, and the collected lung-metastasis samples were weighed by an electronic balance and the surface nodules were counted. RESULTS: We established an improved phytohemagglutinin-polyethylene glycol fusion method and successfully obtained stable melanoma tumor-tumor cell fusion hybrids. Cell size, DNA content, and chromosome numbers of the fusion hybrids were approximately twice those of the parents. The metastatic potential of the fusion hybrids was dramatically enhanced, in contrast to their proliferation rate. Their metastasis was specific to the lungs. CONCLUSIONS: We developed a highly efficient cell fusion method that can be applied in many fields, particularly cancer research. Our study has proven that tumor-tumor cell fusion hybrids in melanoma can acquire enhanced and specific metastatic potential. Thus, blockage of cell fusion may be a new strategy for melanoma metastasis therapy.

Detection of SRS19-6MuLV in mouse dendritic cell sarcoma and its tumorigenesis.

SRS19-6MuLV is a member of the MuLV family originally isolated from the Tianjin-Shanghai-Zunyi complex of murine leukemia. A notable characteristic of this virus is that it induces tumors of multiple hematopoietic lineages, including myeloid, erythroid, T-lymphoid and B-lymphoid. In a previous study, a sequence with high homology to SRS19-6MuLV in a murine dendritic cell sarcoma (DCS) was identified through cDNA expression screening with mAb 983D4. To investigate the relationship between SRS19-6MuLV and DCS, the existence of a specific SRS19-6MuLV DNA fragment in DCS cells, 15 murine tumor cells, 2 murine tumor tissues, 12 normal murine cells/tissues, 11 human tumor cell lines and SRSV/3T3 (NIH/3T3 cells infected with SRS cell supernatant) was detected by PCR. The specific fragment of SRS19-6MuLV was detected in DCS, mouse fore-gastric cancer cells, LⅡ tumor tissue from which DCS is derived and SRSV/3T3. In addition, the integration sites of SRS19-6MuLV in the positive cells were examined by inverse PCR. Thus, 7 integration sites for SRS19-6MuLV were detected in DCS and 3 in SRSV/3T3. Analysis of sequences by BLAST revealed that some of the integration sites were associated with common fragile sites and some Ras-regulating miRNAs. Our results indicate that SRS19-6MuLV not only induced four types of leukemia, but also induced DCS. This virus does not infect human cells. Multiple integration of SRS19-6MuLV into chromosomes around fragile sites accounts for its carcinogenic effects.

[Effect of forced E-cadherin expression on adhesion and proliferation of human breast carcinoma cells].

OBJECTIVE: To investigate the role that E-cadherin (E-cad) plays on cell adhesion and proliferation of human breast carcinoma. METHODS: E-cad expression vector was transfected into an E-cad-negative human breast carcinoma MDA-MB-231 cells. G418 was used to screen positive clones. E-cad, ß-catenin (ß-cat) and cyclin D1 expressions of these clones were confirmed by Western blot. Their cell-cell and cell-matrix adhesion abilities were detected. E-cad/ß-catenin interaction was confirmed by immunoprecipitation. Cell proliferation was evaluated by MTT. Cell apoptosis was analyzed by flow cytometry. Direct two-step immunocytochemistry was used to detect the localization of ß-cat. RESULT: E-cad(+) cell strains Ecad-231-7 and Ecad-231-9 were established. When cultured in ultra-low-binding dishes Ecad-231 cells grow in suspension while Ecad-231-7 and Ecad-231-9 cells grow in large clamps. When co-cultured with HCT116 cells, the average adhesion rates at 30 min are 39.0%, 60.0% and 59.5% for MDA-MB-231, Ecad-231-7 and Ecad-231-9 respectively. The average detachment rates by EDTA for 5 min are 37.4%, 4.2% and 7.4% respectively. So E-cad expression enhanced hemotypic and heterotypic cell-cell adhesion and cell-matrix adhesion. Forced exogenously expressed E-cad could combine with endogenous ß-cat, whereas down stream cyclin D1 expression was significantly decreased, as evidenced by Western blot. The rates of cell apoptosis of MDA-MB-231, Ecad-231-7 and Ecad-231-9 were 1.8%, 2.0% and 2.1%. Expression of E-cad had no obvious effect on the apoptosis of tumor cells with regular culture. ß-cat increased in the cytoplasma. CONCLUSIONS: Two monoclonal tumor cell strains (Ecad-231-7 and Ecad-231-9) stably expressing E-cad were successfully established. E-cad could enhance adhesion and inhibit proliferation of human breast carcinoma cells through a pathway involving ß-cat and cyclin D1.

Expression of CD133 correlates with differentiation of human colon cancer cells.

CD133 has been identified as a cancer stem cell marker in colon and several other cancers, but its function is still unknown. We examined the CD133 expression in 44 human cancer cell lines, and found five of the 8 positive lines were from colon cancer. The CD133 positive subpopulation of colon cancer cells showed more vigorous growth and lower differentiation. Induction of differentiation reduced the CD133-positive population. Knockdown of CD133 expression in colon cancer cells could not induce cellular differentiation. Care must be taken if CD133 is used as the only marker of cancer stem cells in colon cancer, especially in established cell lines. CD133 negatively correlates with cell differentiation, but it is not a regulator of differentiation.